Luminescence quenchers and fluorogenic probes for detection of reactive species

ABSTRACT

Provided herein are compounds or fluorogenic probes which can be used as reagents for measuring, detecting and/or screening ROS or RNS such as peroxynitrite or hypochlorite. Provided also herein are methods that can be used to measure, directly or indirectly, the amount of peroxynitrite or hypochlorite in chemical samples and biological samples such as cells and tissues in living organisms. Specifically, the methods include the steps of contacting the fluorogenic probes disclosed herein with the samples to form one or more fluorescent compounds, and measuring fluorescence properties of the fluorescent compounds. Provided also herein are high-throughput screening fluorescent methods for detecting or screening peroxynitrite or compounds that can increase or decrease the level of peroxynitrite or hypochlorite in chemical and biological samples.

PRIOR RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/042,720, filed Apr. 5, 2008, which is incorporated herein byreference in its entirety.

FIELD

Provided herein are aromatic amine compounds which can be used asluminescence quenchers and/or fluorogenic probes for measuring,detecting or screening reactive nitrogen species (RNS) such asperoxynitrite or reactive oxygen species (ROS) such as hypochlorite.Also provided herein are methods of making the aromatic amine compoundsand methods of using the aromatic amine compounds.

BACKGROUND

Luminescence is generally the emission of light that does not deriveenergy from the temperature of the emitting body. Luminescence may becaused by chemical, biochemical, or crystallographic changes, themotions of subatomic particles, or radiation-induced excitation of anatomic or molecular system. Luminescence quenching refers to any processwhich can decrease the luminescence intensity of a given luminophore. Avariety of processes can result in luminescence quenching, such asexcited state reactions, energy transfer, complex formation andcollisional quenching.

The luminescence quenching process, especially the fluorescencequenching process, through energy transfer has been well studied. When afirst fluorophore is excited and transfers its absorbed energy to asecond fluorophore, the energy transfer results in fluorescent signal atthe emission wavelength of the second fluorophore. However, where thesecond fluorophore shows no fluorescence, the absorbed energy does notresult in fluorescence emission, and the first fluorophore is said to be“quenched”. Similarly, energy transfer can also be utilized to quenchthe emission of other luminescent donors such as phosphorescent andchemiluminescent donors.

The use of a variety of dyes containing at least a luminophore to quenchluminescence such as fluorescence is known in the art. The applicationof luminescence quenching to analyze biological systems is alsowell-studied. However, there is always a need for luminescence quenchershaving different absorption properties to meet the various requirementsof new advances in this field.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) aregenerally known to scientists as very small inorganic or organicmolecules with high reactivity. There are various forms of ROS and RNSincluding free radicals such as superoxide radical, hydroxyl radical,nitric oxide, nitrogen dioxide and organic peroxyl radical as well asnon-radical species such as hydrogen peroxide, singlet oxygen, ozone,nitrous acid, peroxynitrite and hypochlorite. ROS and RNS are theby-products of cellular respiration. Under normal conditions ROS and RNSare present in very low levels and play important roles in cellsignaling, while during oxidative stresses, ROS and RNS levels increasedramatically, which can cause serious damages to various biologicalmolecules such as protein, lipids and DNA. The excessive generation ofROS and RNS has been implicated in a lot of human diseases, such ascardiovascular diseases, inflammatory diseases, metabolic diseases,cancer and central nervous system diseases. Therefore, there is a strongneed for chemicals that can sensitively and selectively measure, detector screen certain ROS and RNS to address their physiological roles bothin vitro and in vivo.

Peroxynitrite and hypochlorite have the strongest oxidizing power amongthe various forms of ROS and RNS, and their selective detections arehighly desirable to clearly explain their critical roles in livingorganisms. Peroxynitrite (ONOO⁻) is a short-lived oxidant species thatis formed in vivo by the diffusion-controlled reaction (k=0.4−1.9×10¹⁰M⁻¹s⁻¹) of nitric oxide (NO) and superoxide (O₂ ^(•−)) in one to onestoichiometry. The oxidant reactivity of peroxynitrite is highlypH-dependent and both peroxynitrite anion and its protonated formperoxynitrous acid can participate directly in one- and two-electronoxidation reactions with biomolecules. The pathological activity ofONOO⁻ is also related to its reaction with the biologically ubiquitousCO₂, thereby producing the highly reactive radicals CO₃ ^(−•) and NO₂^(•) in about 35% yield. As a result of this, peroxynitrite can nitratetyrosine and oxidize proteins, lipids and iron and sulfur clusters ofbiological molecules. Like other oxidizing agents in living organisms,peroxynitrite and its protonated form have been associated with bothbeneficial and harmful effects. However, several studies have implicatedthat peroxynitrite contributes to tissue injury in a number of humandiseases such as ischemic reperfusion injury, rheumatoid arthritis,septic shock, multiple sclerosis, atherosclerosis, stroke, inflammatorybowl disease, cancer, and several neurodegenerative diseases(MacMillan-Crow, L. A. et al., Proc. Natl. Acad. Sci. USA 1996, 93,11853-11858; Rodenas, J. et al., Free Radical. Biol. & Med. 2000, 28,374; Cuzzocrea, S. et al., Pharmacol Rev. 2001, 53, 135-159; Szabo, C.Toxicol. Lett. 2003, 140, 105-112; White, C. R. et al., Proc. Natl.Acad. Sci. USA 1994, 91, 1044-1048; Lipton, S. A. et al., Nature 1993,364, 626-632; Pappolla, M. A. et al., J. Neural Transm. 2000, 107,203-231; Beal, M. F., Free Radical Biol. & Med. 2002, 32, 797-803).

On the other hand, hypochlorite is produced in vivo from hydrogenperoxide and chlorine ions in a chemical reaction catalyzed by theenzyme myeloperoxidase (MPO), which may be secreted by activatedphagocytes in zones of inflammation. As a nucleophilic non-radicaloxidant, hypochlorite can be used as a microbicidal agent (Thomas, E.L., Infect. Immun., 1979, 23, 522-53 1). Furthermore, neither bacterianor normal healthy cells can neutralize its toxic effect because theylack the enzymes required for its catalytic detoxification (Lapenna, D.and Cuccurullo, F., Gen. Pharmacol., 1996, 27, 1145-1147).

Generally, hypochlorite can react with some proteins that may playimportant roles in killing bacterial cells and/or human diseases(Thomas, E. L., Infect. Immun., 1979, 23, 522-531; McKenna, S. M. andDavies, K. J. A., Biochem. J., 1988, 254, 685-692; Hazell, L. J. andStocker, R., Biochem. J., 1993, 290, 165-172; Hazell, L. J., van denBerg, J. J. and Stocker, R., Biochem. J., 1994, 302, 297-304). Whencontacting with proteins, hypochlorite may cause damages to theproteins. For example, hypochlorite may alter protein structures, and/orcause fragmentation and dimerization of proteins. As a strong oxidant,hypochlorite can also oxidize low-density lipoproteins (LDL) rapidly.Furthermore, the reaction of hypochlorite with DNA can also result inboth chemical modifications and structural changes in DNA (Hawkins, C.L. and Davies, M. J., Chem. Res. Toxicol., 2002, 15, 83-92; Prutz, W.A., Arch. Biochem. Biophys. 1996, 332, 110-120; Arch. Biochem. Biophys.1998, 349, 183-191; Arch. Biochem. Biophys. 1999, 371, 107-114).

Because of the above-mentioned uses and roles of ROS and RNS, there is aneed for methods that detect, measure and/or screen ROS such ashypochlorite and/or RNS such as peroxynitrite, including in vivodetection and measurement.

SUMMARY

Provided herein are aromatic amine compounds that can be used asluminescence quenchers and/or fluorogenic probes for measuring,detecting or screening reactive nitrogen species (RNS) or reactiveoxygen species (ROS) such as ¹O₂, O₂ ^(•−), NO, H₂O₂, .OH, ⁻OCl, ONOO⁻and alkylperoxyl radical (ROO^(•)).

In one aspect, the aromatic amine compounds can be represented byformula (I):

-   -   wherein R¹ is hydrogen, alkyl, halogenated alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl,        cycloalkyl, cycloalkenyl or cycloalkynyl;    -   L has one of formulae (II)-(VI)

or a tautomer thereof,

-   -   wherein Y is O-A, S-A or NR²R³;    -   each of Y¹, Y², Y³ and Y⁴ is independently O, S, NR^(2′)R^(3′)        or N⁺R^(2′)R^(3′);    -   V is N or CR″;    -   each of R², R³, R^(2′) and R^(3′) is independently H, alkyl,        halogenated alkyl, alkenyl, alkynyl, alkoxyalkyl, heteroalkyl,        cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,        aminoalkyl, aryl, alkaryl, arylalkyl, alkyloxy, carboxyalkyl,        alkylamido, alkoxyamido, sulfonylaryl or acyl;    -   each of R, R′, R″, R^(a) and R^(b) is independently H, CN,        alkyl, halogenated alkyl, alkenyl, alkynyl, alkoxyalkyl,        heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,        heterocyclyl, aminoalkyl, aryl, alkaryl, arylalkyl, alkyloxy,        carboxyalkyl, alkylamino, alkoxyamino, alkylamido, alkoxyamido,        sulfonylaryl or acyl;    -   A is H, alkyl, alkenyl, alkynyl, alkoxyalkyl, heteroalkyl,        cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,        aminoalkyl, aryl, alkaryl, arylalkyl, carboxyalkyl,        alkoxycarbonyl, acyl or aminocarbonyl;    -   each of K¹-K⁵² is independently H, halo, alkyl, halogenated        alkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl,        heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,        hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino,        dialkylamino, alkylarylamino, diarylamino, acylamino, hydroxy,        thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,        arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,        trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl,        sulfonate ester, phosphate ester, —C(═O)—P¹ or —C(═O)—Z—P²;    -   each of P¹ and P² is independently hydrogen, halo, alkoxy,        hydroxy, thio, alkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate,        amino, alkylamino, arylamino, dialkylamino, alkylarylamino,        diarylamino, alkylthio, heteroalkyl, or heterocyclyl having from        3 to 7 ring atoms; and    -   Z is alkylene, alkenylene, alkynylene, arylene, aralkylene or        alkarylene; and    -   Q is substituted or unsubstituted phenyl having formula (VIIa):

-   -   wherein each of R⁴, R⁵, R⁶, R⁷ and R⁸ is independently H, alkyl,        alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl,        cycloalkynyl, aryl, alkylaryl, arylalkyl, heterocyclyl, hydroxy,        alkoxy, alkoxyalkyl, alkoxyalkoxy, acyl, alkylcarbonylalkyl,        halogentaed alkylcarbonylalkyl such as        trifluoromethylcarbonylalkyl, aminoalkyl, carboxyalkyl,        alkoxycarbonyl, alkoxycarbonylalkyl, aminocarbonyl, or NR⁹R¹⁰ or        R⁴ and R⁵ together, R⁵ and R⁶ together, R⁶ and R⁷ together or R⁷        and R⁸ together forming a 5- or 6-membered cycloalkyl,        heterocyclyl, aryl or heteroaryl ring fused with the phenyl ring        of formula (VIIa); and    -   each of R⁹ and R¹⁰ is independently H, alkyl, alkenyl, alkynyl,        alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl,        or polyether;    -   with the proviso that when L has formula (II) where Y is NR²R³,        then R⁶ of Q is hydroxy, alkenyl, alkynyl, heteroalkyl,        cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclyl,        alkoxyalkyl, alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogentaed        alkylcarbonylalkyl such as trifluoromethylcarbonylalkyl,        carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, aminocarbonyl        or NR⁹R¹⁰ or R⁴ and R⁵ together, R⁵ and R⁶ together, R⁶ and R⁷        together or R⁷ and R⁸ together form a 5- or 6-membered        cycloalkyl, heterocyclyl, aryl or heteroaryl ring fused with the        phenyl ring of formula (VIIa).

In some embodiments, each of R, R′, R″, R^(a) and R^(b) independentlyhas formula (VII):

wherein each of R^(4′), R^(5′), R^(6′), R^(7′) and R^(8′) isindependently H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, alkylaryl, arylalkyl, heterocyclyl,hydroxy, alkoxy, alkoxyalkyl, alkoxyalkoxy, acyl, alkylcarbonylalkyl,halogentaed alkylcarbonylalkyl, aminoalkyl, carboxyalkyl,alkoxycarbonyl, alkoxycarbonylalkyl, aminocarbonyl, or NR⁹R¹⁰, or R^(4′)and R^(5′) together, R^(5′) and R^(6′) together, R^(6′) and R^(7′)together or R^(7′) and R^(8′) together forming a 5- or 6-memberedcycloalkyl, heterocyclyl, aryl or heteroaryl ring fused with the phenylring of formula (VII).

In certain embodiments, R^(4′), R^(5′), R^(6′) and R^(7′) of formula(VII) is independently H; and R^(8′) is —COOH, —COR¹⁷, —COOR¹⁸, or—CONR¹⁹R²⁰, wherein R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently H, alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,aryl, alkylaryl, arylalkyl, heterocyclyl, hydroxy, alkoxy, alkoxyalkyl,alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogentaed alkylcarbonylalkyl,aminoalkyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl,aminocarbonyl, or N, R¹⁹ and R²⁰ together forming a 5-or 6-memberedheterocycle having at least a nitrogen atom. In other embodiments,R^(8′) is —CONR¹⁹R²⁰ and N, R¹⁹ and R²⁰ together form a 5- or 6-saturated heterocycle. In further embodiments, the heterocycle issubstituted or unsubstituted piperidine, morpholine, pyrrolidine,oxazolidine, thiomorpholine, thiazolidine or piperazine.

In some embodiments, each of R^(4′), R^(5′), R^(6′) and R^(7′) offormula (VII) is independently H; and R^(8′) is methyl, methoxy, or thelike to make the benzene ring out of the xanthenes ring plane.

In some embodiments, L has formula (II) or a tautomer thereof. In otherembodiments, Y of formula (II) is NR²R³. In other embodiments, Y offormula (II) is OH, OAc or OCH₂OCOCH₃. In further embodiments, each ofK¹, K³, K⁴, K⁶, K⁷, K⁸, K⁹ and K¹⁰ is H; and each of K² and K⁵isindependently H or halo. In still further embodiments, each of K¹, K²,K³, K⁴, K⁵, K⁶, K⁷, K⁸, K⁹ and K¹⁰ is H.

In certain embodiments, L has formula (III) or a tautomer thereof. Inother embodiments, each of K¹¹, K¹², K¹³, K¹⁴, K¹⁶ and K¹⁷ is H; and Kis H or halo.

In some embodiments, L has formula (IV) or (IVa). In other embodiments,each of K¹⁸ and K²⁰ is H; and K¹⁹ is H or halo.

In certain embodiments, L has formula (V). In other embodiments, V is N.In other embodiments, V is CR″. In further embodiments, Y¹ isN⁺R^(2′)R^(3′). In still further embodiments, Y¹ is O. In still furtherembodiments, each of K²¹, K²², K²³, K²⁴, K²⁵ and K²⁶ is H.

In some embodiments, L has formula (VI). In other embodiments, Y² isN⁺R^(2′)R^(3′). In further embodiments, Y² is O. In still furtherembodiments, each of K²⁷-K³⁶ is H.

In some embodiments, L has formula (XX). In other embodiments, Y³ isN⁺R^(2′)R^(3′). In further embodiments, Y³ is O. In still furtherembodiments, each of K³⁷-K⁴⁴ is H. In still further embodiments, each ofK³⁸-K⁴⁴ is H; and K³⁷ is Cl or F.

In some embodiments, L has formula (XXI). In other embodiments, Y⁴ isN⁺R^(2′)R^(3′). In further embodiments, Y⁴ is O. In still furtherembodiments, each of K⁴⁶-K⁵¹ is H, and at least one of K⁴⁵ and K⁵² isindependently Cl or F.

In certain embodiments, R⁶ of formula (VIa) is —OCH₂OCH₃, OH, NR⁹R¹⁰,—CH₂CH₂C(═O)CF₃, or —CH₂CH₂C(═O)OCH₃, wherein each of R⁹ and R¹⁰ isindependently H or alkyl; and each of R⁴, R⁵, R⁷ and R⁸ is H. In otherembodiments, R⁶ is OH, NH₂ or —CH₂CH₂C(═O)CF₃.

In some embodiments, R¹ of formula (I) is H, alkyl, halogenated alkyl,heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl,cycloalkyl, cycloalkenyl, and cycloalkynyl; each of R⁴, R⁵, R⁶, R⁷ andR⁸ is independently H, halogen, alkyl, alkoxy, or polyether; R⁶ is OR¹¹or CH₂CH₂COR¹², where R¹¹ is H, alkyl, alkoxyalkyl, alkanoyl, orpolyether; R¹² is an electron-withdrawing group selected from CF₃,halogen-substituted lower alkyl, or (C═O)—O—V²; and V² is a groupselected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, alkaryl or arylalkyl.

Provided also herein are fluorogenic probe compositions for measuring,detecting or screening peroxynitrite comprising the aromatic aminecompound disclosed herein. In certain embodiments, the aromatic aminecompound is Compound (10), Compound (12), Compound (12a), Compound (22),or Compound (30):

or a tautomer thereof, or a combination thereof.

Provided also herein are fluorogenic probe compositions for measuring,detecting or screening hypochlorite comprising the aromatic aminecompound disclosed herein. In certain embodiments, the aromatic aminecompound is Compound (14):

or a tautomer thereof.

In certain embodiments, the fluorogenic probe compositions disclosedherein further comprise a solvent, an acid, a base, a buffer solution ora combination thereof.

Provided also herein are fluorogenic probe compositions for measuringperoxynitrite or hypochlorite in a sample, wherein the compositionscomprise the aromatic amine compounds disclosed herein. In someembodiments, the fluorogenic probe compositions further comprise asolvent, an acid, a base, a buffer solution or a combination thereof.

Provided also herein are methods for measuring peroxynitrite orhypochlorite in a sample, wherein the methods comprise the steps of:

a) contacting an aromatic amine compound disclosed herein with thesample to form a fluorescent compound; and

b) measuring fluorescence properties of the fluorescent compound todetermine the amount of peroxynitrite or hypochlorite in the sample.

In some embodiments, the sample is a chemical sample or biologicalsample. In other embodiments, the sample is a biological samplecomprising a microorganism, or a cell or tissue from animals.

Provided also herein are high-throughput screening fluorescent methodsfor detecting peroxynitrite or hypochlorite in samples, wherein thehigh-throughput methods comprise the steps of:

a) contacting an aromatic amine compound disclosed herein with thesamples to form one or more fluorescent compounds; and

b) measuring fluorescence properties of the fluorescent compounds todetermine the amount of peroxynitrite or hypochlorite in the samples.

Provided also herein are high-throughput methods for screening one ormore target compounds that can increase or decrease the level ofperoxynitrite or hypochlorite, wherein the high-throughput methodscomprise the steps of:

a) contacting an aromatic amine compound disclosed herein with thetarget compounds to form one or more fluorescent compounds; and

b) measuring fluorescence properties of the fluorescent compounds todetermine the target compounds qualitatively or quantitatively.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts fluorescence spectra showing fluorescence intensities of10 μM of Compound 10 in response to different concentrations of ONOO⁻ atdifferent wavelengths. The spectra were acquired in 0.1 M potassiumphosphate buffer at pH 7.4 where 0.1% DMF was used as a cosolvent andλex was at 520 nm.

FIG. 2 depicts a linear correlation between fluorescence intensity of 10μM of Compound 10 and the concentration of ONOO⁻ measured at 540 nm.

FIG. 3 depicts the fluorescence intensity of 10 μM of Compound 10 invarious ROS/RNS generating systems at 25° C. for 30 minutes measured at540 nm. The concentration of ¹O₂, O₂ ^(•−), NO, ROO. and H₂O₂concentration was 100 μM. The concentration of .OH, ⁻OCl and ONOO⁻concentration was 10 μM.

FIG. 4 depicts fluorescence spectra showing fluorescence intensities of1 μM of Compound 12 in response to different concentrations of ONOO⁻ atdifferent wavelengths. The spectra were acquired in 0.1 M potassiumphosphate buffer at pH 7.4 where 0.1% DMF was used as a cosolvent.

FIG. 5 depicts the fluorescence intensity of 1 μM of Compound 12 invarious ROS/RNS generating systems at 25° C. for 30 minutes measured at530 nm. The concentration of each of ¹O₂, O₂ ^(•−), NO, ROO. and H₂O₂was10 μM. The concentration each of .OH, ⁻OCl and ONOO⁻ was 1 μM.

FIG. 6 depicts fluorescence spectra showing fluorescence intensities of1 μM of Compound 14 in response to different concentrations of ⁻OCl atdifferent wavelengths. The spectra were acquired in 0.1 M potassiumphosphate buffer at pH 7.4 where 0.1% DMF was used as a cosolvent.

FIG. 7 depicts the fluorescence intensity of 1 μM of Compound 14 invarious ROS/RNS generating systems at 25° C. for 30 minutes measured at530 nm. The concentration of each of ¹O₂, O₂ ^(•−), NO, ROO., H₂O₂, .OH,⁻OCl and ONOO⁻ was 5 μM.

FIG. 8 shows fluorescent microscopy results of Murine J744.1 macrophagesunder different stimulation conditions. The macrophage cells wereincubated with Compound 10 at a concentration of 20 μM. The macrophagesin (A) were the Control. The macrophages in (B) were stimulated with LPSand IFN-γ for 4 hours. The macrophages in (C) were stimulated with LPSand IFN-γ for 4 hours, followed by further stimulation with PMA for 0.5hours.

FIG. 9 shows fluorescent microscopy results of Murine J744.1 macrophagesunder different stimulation conditions. The macrophage cells wereincubated with Compound 12 and MitoTracker Red CMXRos (purchased fromInvitrogen) at concentrations of 20 μM. The macrophages in (A)-(B) werethe control. The macrophages in (C)-(F) were stimulated with LPS.

FIG. 10 shows two-photon fluorescent microscopy results of Murine J744.1macrophages under stimulation conditions. The macrophage cells wereincubated with Compound 12a at a concentration of 20 μM. The macrophagesin (A) were the control. The macrophages in (B) were stimulated with LPSfor 4 hours.

FIG. 11 depicts fluorescence spectra showing fluorescence intensities of10 μM of Compound 30 in response to different concentrations ofperoxynitrite at different wavelengths. The spectra were acquired withexcitation at 520 nm in 0.1 M potassium phosphate buffer at pH 7.4 where0.1% DMF was used as a cosolvent.

DEFINITIONS

To facilitate the understanding of the subject matter disclosed herein,a number of terms, abbreviations or other shorthand as used herein aredefined below. Any term, abbreviation or shorthand not defined isunderstood to have the ordinary meaning used by a skilled artisancontemporaneous with the submission of this application.

“Amino” refers to a primary, secondary, or tertiary amine which may beoptionally substituted. Specifically included are secondary or tertiaryamine nitrogen atoms which are members of a heterocyclic ring. Alsospecifically included, for example, are secondary or tertiary aminogroups substituted by an acyl moiety. Some non-limiting examples ofamino group include —NR′R″ wherein each of R′ and R″ is independently H,alkyl, aryl, aralkyl, alkaryl, cycloalkyl, acyl, heteroalkyl, heteroarylor heterocycyl.

“Alkyl” refers to a fully saturated acyclic monovalent radicalcontaining carbon and hydrogen, and which may be branched or a straightchain. In some embodiments, alkyl contains from about 1 to about 25carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, n-heptyl, n-hexyl, n-octyl, and n-decyl.“Lower alkyl” refers to an alkyl radical of one to six carbon atoms, asexemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl,n-pentyl, and isopentyl.

“Heteroalkyl” refers to an alkyl group having one or more of the carbonatoms within the alkyl group substituted by a heteroatom such as O, Sand N. In some embodiments, the heteroalkyl group comprises one or moreO atoms. In other embodiments, the heteroalkyl group comprises one ormore S atoms. In further embodiments, the heteroalkyl group comprisesone or more aminylene groups. In certain embodiments, the heteroalkylgroup comprises two or more O, S, aminylene or a combination thereof.

“Alkenyl” or “alkenylene” respectively refers to a monovalent ordivalent hydrocarbyl radical which has at least one double bond. Thealkenyl or alkenylene group may be cyclic, branched acyclic or straightacyclic. In some embodiments, the alkenyl or alkenylene group containsonly one double bond. In other embodiments, the alkenyl or alkenylenegroup contains two or more double bonds. In further embodiments, thealkenyl or alkenylene group can be a lower alkenyl or alkenylenecontaining from two to eight carbon atoms in the principal chain. Infurther embodiments, the alkenyl or alkenylene group can have one doublebond and up to 25 carbon atoms, as exemplified by ethenyl, propenyl,isopropenyl, butenyl, isobutenyl, hexenyl, and the like.

“Alkynyl” or “alkynylene” respectively refers to a monovalent ordivalent hydrocarbyl radical which has at least a triple bond. In someembodiments, the alkynyl or alkynylene group contains only one triplebond. In other embodiments, the alkynyl or alkynylene group contains twoor more triple bonds. In further embodiments, the alkynyl or alkynylenegroup can be a lower alkynyl or alkynylene containing from two to eightcarbon atoms in the principal chain. In further embodiments, the alkynylor alkynylene group can have one triple bond and up to 20 carbon atoms,as exemplified by ethynyl, propynyl, isopropynyl, butynyl, isobutynyl,hexynyl, and the like.

“Aromatic” or “aromatic group” refers to aryl or heteroaryl.

“Aryl” refers to optionally substituted carbocyclic aromatic groups. Insome embodiments, the aryl group includes a monocyclic or bicyclic groupcontaining from 6 to 12 carbon atoms in the ring portion, such asphenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl orsubstituted naphthyl. In other embodiments, the aryl group is phenyl orsubstituted phenyl.

“Aralkyl” refers to an alkyl group which is substituted with an arylgroup. Some non-limiting examples of aralkyl include benzyl andphenethyl.

“Alkaryl” refers to an aryl group which is substituted with an alkylgroup. Some non-limiting examples of alkaryl include methylphenyl andmethylnaphthyl.

“Acyl” refers to a monovalent group of the formula —C(═O)H,—C(═O)-alkyl, —C(═O)-aryl, —C(═O)-aralkyl, or —C(═O)-alkaryl.

“Halogen” refers to fluorine, chlorine, bromine and iodine.

“Halo” refers to fluoro, chloro, bromo and iodo.

“Heteroatom” refers to atoms other than carbon and hydrogen.

“Heterocyclo” or “heterocyclyl” refers to optionally substituted, fullysaturated or unsaturated, monocyclic or bicyclic, aromatic ornonaromatic groups having at least one heteroatom, such as O, S, N, Band P, in at least one ring. The aromatic heterocyclyl (i.e.,heteroaryl) group can have 1 or 2 oxygen atoms, 1 or 2 sulfur atoms,and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to theremainder of the molecule through a carbon or heteroatom. Somenon-limiting examples of heteroaryl include furyl, thienyl, thiazolyl,pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl andthe like.

“Hydrocarbon” or “hydrocarbyl” refers to organic compounds or radicalsconsisting exclusively of the elements carbon and hydrogen. Hydrocarbylincludes alkyl, alkenyl, alkynyl, and aryl moieties. Hydrocarbyl alsoincludes alkyl, alkenyl, alkynyl, and aryl moieties substituted withother aliphatic, cyclic or aryl hydrocarbon groups, such as alkaryl,alkenaryl and alkynaryl. In some embodiments, “hydrocarbon” or“hydrocarbyl” comprises 1 to 30 carbon atoms.

“Hydrocarbylene” refers to a divalent group formed by removing twohydrogen atoms from a hydrocarbon, the free valencies of which are notengaged in a double bond, e.g. arylene, alkylene, alkenylene,alkynylene, aralkylene or alkarylene.

“Substituted” as used herein to describe a compound or chemical moietyrefers to that at least one hydrogen atom of that compound or chemicalmoiety is replaced with a second chemical moiety. Non-limiting examplesof substituents are those found in the exemplary compounds andembodiments disclosed herein, as well as halogen; alkyl; heteroalkyl;alkenyl; alkynyl; aryl, heteroaryl, hydroxy; alkoxyl; amino; nitro;thiol; thioether; imine; cyano; amido; phosphonato; phosphine; carboxyl;thiocarbonyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; oxo;haloalkyl (e.g., trifluoromethyl); carbocyclic cycloalkyl, which can bemonocyclic or fused or non-fused polycyclic (e.g., cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl) or a heterocycloalkyl, which canbe monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl or thiazinyl); carbocyclic orheterocyclic, monocyclic or fused or non-fused polycyclic aryl (e.g.,phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl,pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl,pyrimidinyl, benzimidazolyl, benzothiophenyl or benzofuranyl); amino(primary, secondary or tertiary); o-lower alkyl; o-aryl, aryl;aryl-lower alkyl; —CO₂CH₃; —CONH₂; —OCH₂CONH₂; —NH₂; —SO₂NH₂; —OCHF₂;—CF₃; —OCF₃; —NH(alkyl); —N(alkyl)₂; —NH(aryl); —N(alkyl)(aryl);—N(aryl)₂; —CHO; —CO(alkyl); —CO(aryl); —CO₂(alkyl); and —CO₂(aryl); andsuch moieties can also be optionally substituted by a fused-ringstructure or bridge, for example —OCH₂O—. These substituents canoptionally be further substituted with a substituent selected from suchgroups. All chemical groups disclosed herein can be substituted, unlessit is specified otherwise. For example, “substituted” alkyl, alkenyl,alkynyl, aryl, hydrocarbyl or heterocyclo moieties described herein aremoieties which are substituted with a hydrocarbyl moiety, a substitutedhydrocarbyl moiety, a heteroatom, or a heterocyclo. Further,substituents may include moieties in which a carbon atom is substitutedwith a heteroatom such as nitrogen, oxygen, silicon, phosphorus, boron,sulfur, or a halogen atom. These substituents may include halogen,heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protectedhydroxy, keto, acyl, acyloxy, nitro, amino, amido, cyano, thiol, ketals,acetals, esters and ethers.

“Luminescence” refers to the emission of light that does not deriveenergy from the temperature of the emitting body. Luminescence may becaused by chemical, biochemical, or crystallographic changes, themotions of subatomic particles, or radiation-induced excitation of anatomic system. Luminescence includes, but is not limited to,phosphorescence, fluorescence, chemoluminescence, bioluminescence,crystalloluminescence, electroluminescence such as cathodoluminescence,mechanoluminescence, such as sonoluminescence, triboluminescence,fractoluminescence and piezoluminescence, photoluminescence such asphosphorescence and fluorescence, radioluminescence, andthermoluminescence.

“Luminophore” refers to an atom or a chemical group in a chemicalcompound that manifests luminescence. There are organic and inorganicluminophores. In some embodiments, the luminophores disclosed herein areorganic luminophores such as rhodol, rhodamine, resorufin or aderivative thereof. Luminophores includes, but is not limited to,phosphores, fluorophores, chemolumiphores, biolumiphores,crystallolumiphores, electrolumiphores such as cathodolumiphores,mechanolumiphores, such as sonolumiphores, tribolumiphores,fractolumiphores and piezolumiphores, photolumiphores, radiolumiphores,and thermolumiphores.

“Photoluminescence” refers to a process in which a substance absorbsphotons (electromagnetic radiation) and then radiates photons back out.The substance after absorbing the photons is excited to a higher energystate and then returns to a lower energy state accompanied by theemission of a photon. Two common types of photoluminescence includephosphorescence and fluorescence.

“Luminescence Quencher” refers to a compound that can eliminate,partially or totally, the luminescence of a luminophore.

“Fluorescence” refers to a luminescence where the molecular absorptionof a photon triggers the emission of another photon with a longerwavelength. In some embodiments, the absorbed photon is in theultraviolet range, and the emitted light is in the visible range.

“Fluorophore” refers to a small molecule or a part of a large molecule,that can be excited by light to emit fluorescence. In some embodiments,fluorophores efficiently produce fluorescence upon excitation with lightwhich has a wavelength from about 200 nanometers to about 1000nanometers, or from about 500 nanometers to about 800 nanometers. Theintensity and wavelength of the emitted radiation generally depend onboth the fluorophore and the chemical environment of the fluorophore. Afluorophore may be selected from acridine orange, anthracene ring,allophycocyanin, BODIPY, cyanines, coumarin, Edans, Eosin, Erythrosin,fluorescamine, fluorescein, FAM (carboxyfluorescein), HEX(hexachlorofluorescein), JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy-fluorescein), Oregon Green,phycocyanin, phycoerythrin, rhodamine, ROX (Carboxy-X-rhodamine), TAMRA(carboxytetramethylrhodamine), TET (tetrachloro-fluorescein), Texas red,tetramethylrhodamine, and xanthines. Other non-limiting examples can befound in The Handbook: a Guide to Fluorescent Probes and LabelingTechnologies (10th Edition, Molecular Probes, Eugene, Oreg., 2006),which are incorporated herein by reference.

“Phosphorescence” refers to a specific type of photoluminescence relatedto fluorescence. Unlike fluorescence, a phosphorescent material does notimmediately re-emit the radiation it absorbs. The slower time scales ofthe re-emission are associated with “forbidden” energy state transitionsin quantum mechanics. As these transitions occur less often in certainmaterials, absorbed radiation may be re-emitted at a lower intensity forup to several hours in some embodiments.

“Chemiluminescence” or “chemoluminescence” refers to the effect ofluminescence as the result of a chemical reaction.

“Bioluminescence” refers to the production and emission of light by aliving organism as the result of a chemical reaction during whichchemical energy is converted to light energy.

“Crystalloluminescence” refers to the effect of luminescence producedduring crystallization.

“Electroluminescence” refers to the effect of luminescence where amaterial emits light in response to an electric current passed throughit, or to a strong electric field.

“Cathodoluminescence” refers to the effect of luminescence whereby abeam of electrons is generated by an electron gun (e.g. cathode raytube) and then impacts on a luminescent material such as a phosphor,causing the material to emit visible light.

“Mechanoluminescence” refers to the effect of luminescence as the resultof any mechanical action on a solid. It can be produced throughfriction, ultrasound or other means.

“Triboluminescence” refers to the effect of luminescence in which lightis generated via the breaking of asymmetrical bonds in a crystal whenthat material is scratched, crushed, or rubbed.

“Fractoluminescence” refers to the emission of light from the fractureof a crystal.

“Piezoluminescence” refers to the effect of luminescence caused bypressure that results only in elastic deformation.

“Radioluminescence” refers to the effect of luminescence produced in amaterial by the bombardment of ionizing radiation such as betaparticles.

“Thermoluminescence” refers to the effect of luminescence where somemineral substances store energy when exposed to ultraviolet or otherionising radiation. This energy is released in the form of light whenthe mineral is heated.

“Reactive group” or “Rg” refers to a group that is highly reactivetoward an amine, a thio, an alcohol, an aldehyde or a ketone. Somenon-limiting examples of reactive group include phosphoramidite,succinimidyl ester of a carboxylic acid, haloacetamide, hydrazine,isothiocyanate, maleimide, perfluorobenzamido, azidoperfluorobenzamidoand so on.

“Conjugated substance” or “Cg” refers to a desired substance which needsto be conjugated and generally possess a suitable functional group forcovalent reaction with a respective reactive group, Rg. Somenon-limiting examples of conjugated substances include conjugates ofantigens, steroids, vitamins, drugs, haptens, metabolites, toxins, aminoacids, peptides, nucleotides, oligonucleotides, nucleic acid,carbohydrates, lipids, and the like.

“Reactive oxygen species” or ROS refer to oxygen-containing ions, freeradicals as well as non-radical species. Some non-limiting examples ofreactive oxygen species include ¹O₂, O₂ ^(•−), ROO^(•), ^(•)OH, OCl⁻,and H₂O₂.

“Reactive nitrogen species” or RNS refer to nitrogen-containing ions,free radicals as well as non-radical species. Some non-limiting examplesof reactive nitrogen species include nitric oxide (NO^(•)), nitrogendioxide (NO₂ ^(•)), nitrite (NO₂ ⁻), and peroxynitrite (ONOO).

“Fluorogenic probe” refers to a latent fluorescent molecule, whosefluorescence stays in “off” state before reacting with the target andmay switch to “on” state after reacting with the target. In someembodiments, the fluorogenic probes disclosed herein do not reactsubstantially with reactive oxygen species and reactive nitrogenspecies. In other embodiments, the fluorogenic probes disclosed hereinmay react substantially with reactive oxygen species and reactivenitrogen species.

“Peroxynitrite probe” refers to a compound that can react withperoxynitrite to form a fluorescent compound. In some embodiments, theperoxynitrite probes disclosed herein do not react substantially withperoxynitrite. In other embodiments, the peroxynitrite probes disclosedherein may react substantially with peroxynitrite.

“Hypochlorite probe” refers to a compound that can react withhypochlorite to form a fluorescent compound. In some embodiments, thehypochlorite probes disclosed herein do not react substantially withhypochlorite. In other embodiments, the hypochlorite probes disclosedherein may react substantially with hypochlorite.

“Quinone” refers to a compound comprising a cyclohexadienedione moiety.Some non-limiting examples of quinones include 1,4-benzoquinone,1,2-benzoquinone, 1,4-naphthoquinone, anthraquinone, phenanthraquinone,and the like.

“Reacting”, “adding” or the like refers to contacting one reactant,reagent, solvent, catalyst, reactive group or the like with anotherreactant, reagent, solvent, catalyst, reactive group or the like.Reactants, reagents, solvents, catalysts, reactive group or the like canbe added individually, simultaneously or separately and can be added inany order. They can be added in the presence or absence of heat and canoptionally be added under an inert atmosphere. In some embodiments,“reacting” refers to in situ formation or intra-molecular reaction wherethe reactive groups are in the same molecule.

“Substantially react” refers to that at least a reactant of a reactionis consumed by an amount of more than about 75% by mole, by more thanabout 80% by mole, by more than about 85% by mole, or by more than about90% by mole. In some embodiments, “substantially react” refers to thatthe reactant is consumed by more than about 95% by mole. In otherembodiments, “substantially react” refers to that the reactant isconsumed by more than about 97% by mole. In further embodiments,“substantially react” refers to that the reactant is consumed by morethan about 99% by mole.

“High-throughput method” refers to a method that can autonomouslyprocess or evaluate a large number of samples. In some embodiments,informatics systems can be used and implemented in the high-throughputmethod. The informatics systems can provide the software control of thephysical devices used in the high-throughput method, as well as organizeand store electronic data generated by the high-throughput method.

DETAILED DESCRIPTION

Provided herein are aromatic amine compounds that can be used asluminescence quenchers and/or fluorogenic probes for measuring,detecting or screening reactive nitrogen species (RNS) such asperoxynitrite or reactive oxygen species (ROS) such as hypochlorite. Insome embodiments, the aromatic amine compounds disclosed herein can beused as luminescence quenchers. In other embodiments, the aromatic aminecompounds disclosed herein can be used to detect, measure or screenperoxynitrite or hypochlorite selectively and specifically. In furtherembodiments, the aromatic amine compounds disclosed herein can be usedto selectively react with peroxynitrite or hypochlorite in the presenceof other reactive oxygen and/or nitrogen species such as ¹O₂, O₂ ^(•−),NO, H₂O₂, .OH, ⁻OCl, ONOO⁻ and alkylperoxyl radical (ROO^(•)).

The aromatic amine compounds disclosed herein generally can berepresented by formula (I):

wherein L is a luminophore; Q is a luminescence quenching moiety; and R¹is H, alkyl, halogenated alkyl, alkenyl, alkynyl, alkoxyalkyl,heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,aminoalkyl, aryl, alkaryl, arylalkyl, alkyloxy, carboxyalkyl,alkylamino, alkoxyamino, alkylamido, alkoxyamido, sulfonylaryl or acyl.In some embodiments, both R¹ and Q are luminescence quenching moieties.In other embodiments, R¹ and Q are the same. In further embodiments, R¹and Q are different.

In some embodiments, N, Q and R¹ together form a 4-, 5-, 6-, 7- or8-membered saturated heterocycle containing at least a nitrogen. Inother embodiments, N, Q and R¹ together form a 5- or 6- memberedsaturated heterocycle containing at least a nitrogen. In furtherembodiments, N, Q and R¹ together form a 5- or 6-saturated heterocycleselected from substituted or unsubstituted piperidine, morpholine,pyrrolidine, oxazolidine, thiomorpholine, thiazolidine or piperazine.

Any luminophore that has luminescence properties can be used herein. Incertain embodiments, the luminophore L is a phosphore, fluorophore,chemolumiphore, biolumiphore, crystallolumiphore, electrolumiphore,mechanolumiphore, photolumiphore, radiolumiphore or thermolumiphore. Inother embodiments, the luminophore L is a phosphore, fluorophore orchemolumiphore.

In some embodiments, the luminophore L is a fluorophore group. Somenon-limiting examples of suitable fluorophore groups include monovalentfluorescent groups derived by removing one atom or group, such as H, OHor amino group, from substituted or unsubstituted fluorescein, BODIPY(boron dipyrrimethene), porphyrins, sulforhodamines, acridine orange,acridine yellow, auramine O, euxanthin, luciferin, benzanthrone,9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene,calcein, carboxyfluorescein, 1-chloro-9,10-bis(phenylethynyl)anthracene,coumarins such as 7-hydroxycoumarin, cyanine,4′,6-diamidino-2-phenylindole, ethidium bromide, perylene, phycobilins,phycoerythrin, phycoerythrobilin, rhodol, rhodamine, rubrene, stilbene,Texas Red, naphthofluorescein or a derivative thereof. Othernon-limiting examples of suitable fluorophore groups can be found in TheHandbook: a Guide to Fluorescent Probes and Labeling Technologies (10thEdition, Molecular Probes, Eugene, Oreg., 2006), which is incorporatedherein by reference.

In certain embodiments, the luminophore L is rhodol, rhodamine,resorufin, fluorescein or a derivative thereof. In other embodiments, Lhas one of formulae (II)-(VI):

-   -   or a tautomer thereof,    -   wherein Y is O-A, S-A or NR²R³;    -   each of Y¹, Y², Y³ and Y⁴ is independently O, S, NR^(2′)R^(3′)        or N⁺R^(2′)R^(3′);    -   V is N or CR″;    -   each of R², R³, R^(2′) and R^(3′) is independently H, alkyl,        halogenated alkyl, alkenyl, alkynyl, alkoxyalkyl, heteroalkyl,        cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,        aminoalkyl, aryl, alkaryl, arylalkyl, alkyloxy, carboxyalkyl,        alkylamido, alkoxyamido, sulfonylaryl or acyl;    -   each of R, R′, R″, R^(a) and R^(b) is independently H, CN,        alkyl, halogenated alkyl, alkenyl, alkynyl, alkoxyalkyl,        heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,        heterocyclyl, aminoalkyl, aryl, alkaryl, arylalkyl, alkyloxy,        carboxyalkyl, alkylamino, alkoxyamino, alkylamido, alkoxyamido,        sulfonylaryl or acyl;    -   A is H, alkyl, alkenyl, alkynyl, alkoxyalkyl, heteroalkyl,        cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,        aminoalkyl, aryl, alkaryl, arylalkyl, carboxyalkyl,        alkoxycarbonyl, acyl or aminocarbonyl;    -   each of K¹-K⁵² is independently H, halo, alkyl, halogenated        alkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl,        heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,        hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino,        dialkylamino, alkylarylamino, diarylamino, acylamino, hydroxy,        thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,        arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,        trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl,        sulfonate ester, phosphate ester, —C(═O)—P¹ or —C(═O)—Z—P²;    -   each of P¹ and P² is independently hydrogen, halo, alkoxy,        hydroxy, thio, alkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate,        amino, alkylamino, arylamino, dialkylamino, alkylarylamino,        diarylamino, alkylthio, heteroalkyl, or heterocyclyl having from        3 to 7 ring atoms; and    -   Z is alkylene, alkenylene, alkynylene, arylene, aralkylene or        alkarylene.

In some embodiments, L has formula (II):

or a tautomer thereof,wherein Y and K¹-K¹⁰ are as disclosed herein. In some embodiments, Y isOH, CH₃C(═O)O or NR²R³; each of K¹-K¹⁰ is independently H, halo, alkyl,halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl,alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino,alkylarylamino, diarylamino, acylamino, hydroxy, thio, thioalkyl,alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano,nitro, sulfhydryl, carbamoyl, trifluoromethyl, phenoxy, benzyloxy,sulfonyl, phosphonyl, sulfonate ester, or phosphate ester; and each ofR² and R³ is independently H or alkyl.

In certain embodiments, each of K¹, K³, K⁴, K⁶, K⁷, K⁸, K⁹ and K¹⁰ is H;and each of K² and K⁵ is independently H or halo. In other embodiments,each of K¹-K¹⁰ is H. In further embodiments, Y is OH or CH₃C(═O)O. Instill further embodiments, Y is OH or CH₃C(═O)O; and each of K¹-K¹⁰ isH. In still further embodiments, Y is OH or CH₃C(═O)O; each of K¹, K³,K⁴, K⁶, K⁷, K⁸, K⁹ and K¹⁰ is H; and each of K² and K⁵ is independentlyH or halo.

In some embodiments, Y is NR²R³. In other embodiments, R² together withK² or R³ together with K³ form a part of a 5- or 6-membered saturated orunsaturated ring wherein the ring is optionally substituted. In otherembodiments, R², R³, K¹ and K² together form a part of a saturated orunsaturated bicyclic ring wherein the bicyclic ring is substituted orunsubstituted.

In certain embodiments, the tautomer of formula (II) has formula (IIa):

wherein Y and K¹-K¹⁰ are as disclosed herein. In some embodiments, Y offormula (IIa) is NR²R³.

In other embodiments, the tautomer of formula (II) has formula (IIb):

wherein Y is OH, SH or NHR²; R² and K¹-K¹⁰ are as disclosed herein; andY′ is O, S, or NR² which is derived by removing one hydrogen from thecorresponding Y.

In some embodiments, L has formula (III):

or a tautomer thereof,wherein K¹¹-K¹⁷ are as disclosed herein. In some embodiments, each ofK¹¹-K¹⁷ is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In other embodiments, each of K¹¹, K¹², K¹³,K¹⁴, K¹⁶ and K¹⁷ is H; and K¹⁵ is H or halo.

In certain embodiments, the tautomer of formula (III) has formula(IIIa):

wherein K¹¹-K¹⁷ are as disclosed herein.

In certain embodiments, L has formula (IV):

or a tautomer thereof,wherein R and K¹⁸-K²⁰ are as disclosed herein. In some embodiments, eachof K¹⁸-K²⁰ is independently H, halo, alkyl, halogenated alkyl,heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl,cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino,alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino,acylamino, hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl,aryloxy, arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In other embodiments, each of K¹⁸ and K²⁰ isH; and K¹⁹ is H or halo.

In some embodiments, L has formula (IVa):

or a tautomer thereof,wherein K¹⁸-K²⁰ are as disclosed herein. In other embodiments, each ofK¹⁸ and K²⁰ is H; and K¹⁹ is halo such as F, Cl, Br or I. In furtherembodiments, each of K¹⁸ and K²⁰ is H; and K¹⁹ is Cl.

In some embodiments, L has formula (V):

or a tautomer thereof,wherein Y¹, V and K²¹-K²⁶ are as disclosed herein.

In certain embodiments, L has formula (V) wherein V is N. In otherembodiments, L has formula (Va):

or a tautomer thereof,wherein Y¹ and K²¹-K²⁶ are as disclosed herein. In further embodiments,Y¹ is N⁺R^(2′)R^(3′). In further embodiments, each of K²¹-K²⁶ of formula(Va) is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In still further embodiments, each of K²¹-K²⁶is H.

In certain embodiments, L has formula (V) wherein V is N; and Y¹ isN⁺R^(2′)R^(3′). In other embodiments, L has formula (Vb):

or a tautomer thereof wherein R^(2′), R^(3′) and K²¹-K²⁶ are asdisclosed herein. In further embodiments, each of K²¹-K²⁶ of formula(Vb) is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In still further embodiments, each of K²¹-K²⁶is H.

In some embodiments, N, R^(2′) and R^(3′) of formula (Vb) together forma 5- or 6-membered saturated heterocycle containing at least a nitrogen.In still further embodiments, the 5- or 6-membered saturated heterocycleis substituted or unsubstituted piperidine, morpholine, pyrrolidine,oxazolidine, thiomorpholine, thiazolidine or piperazine. In stillfurther embodiments, one of R^(2′) and R^(3′) is a Q group as disclosedherein. In still further embodiments, both R^(2′) and R^(3′) are a Qgroup and R^(2′) and R^(3′) may be the same or different.

In certain embodiments, R^(2′) together with K²¹ or R^(3′) together withK²² form a part of a 5- or 6-membered saturated or unsaturated ringwherein the ring is optionally substituted. In other embodiments,R^(2′), R^(3′), K²¹ and K²² together form a part of a saturated orunsaturated bicyclic ring wherein the bicyclic ring is substituted orunsubstituted.

In some embodiments, L has formula (V) wherein V is N; and Y¹ is O. Inother embodiment, L has formula (Vc):

or a tautomer thereof,wherein K²¹-K²⁶ are as disclosed herein. In further embodiments, each ofK²¹-K²⁶ of formula (Vc) is independently H, halo, alkyl, halogenatedalkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl,heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl,aminoalkyl, amino, alkylamino, arylamino, dialkylamino, alkylarylamino,diarylamino, acylamino, hydroxy, thio, thioalkyl, alkoxy, alkylthio,alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano, nitro, sulfhydryl,carbamoyl, trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl,sulfonate ester or phosphate ester. In still further embodiments, eachof K²¹-K²⁶ is H.

In some embodiments, L has formula (V) where V is CR″. In otherembodiment, L has formula (Vd):

or a tautomer thereof,wherein R″, Y¹ and K²¹-K²⁶ are as disclosed herein. In furtherembodiments, Y¹ is O or N⁺R^(2′)R^(3′). In further embodiments, each ofK²¹-K²⁶ of formula (Vd) is independently H, halo, alkyl, halogenatedalkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl,heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl,aminoalkyl, amino, alkylamino, arylamino, dialkylamino, alkylarylamino,diarylamino, acylamino, hydroxy, thio, thioalkyl, alkoxy, alkylthio,alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano, nitro, sulfhydryl,carbamoyl, trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl,sulfonate ester or phosphate ester. In still further embodiments, eachof K²¹-K²⁶ is H.

In certain embodiments, K²² and K²³ of formula (Va), (Vb), (Vc) or (Vd)together or K²⁵ and K²⁶ of formula (Va), (Vb), (Vc) or (Vd) togetherform a part of a 5- or 6-membered saturated or unsaturated ring such asa benzo ring wherein the 5- or 6-membered saturated or unsaturated ringis substituted or unsubstituted. In further embodiments, K²² and K²³together or K²⁵ and K²⁶ together form a benzo ring wherein the benzoring is substituted or unsubstituted.

In some embodiments, L has formula (VI):

or a tautomer thereof wherein R′, Y² and K²⁷-K³⁶ are as disclosedherein. In other embodiments, Y² is O or N⁺R^(2′)R^(3′)

In certain embodiments, L has formula (VI) wherein Y² is O. In otherembodiments, L has formula (VIa):

or a tautomer thereof wherein R′ and K²⁷-K³⁶ are as disclosed herein. Infurther embodiments, each of K²⁷-K³⁶ of formula (VIa) is independentlyH, halo, alkyl, halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aryl,aralkyl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino,alkylarylamino, diarylamino, acylamino, hydroxy, thio, thioalkyl,alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano,nitro, sulfhydryl, carbamoyl, trifluoromethyl, phenoxy, benzyloxy,sulfonyl, phosphonyl, sulfonate ester or phosphate ester. In stillfurther embodiments, each of K²⁷-K³⁶ is H.

In certain embodiments, L has formula (VI) wherein Y² is N⁺R^(2′)R^(3′).In other embodiments, L has formula (VIb):

or a tautomer thereof wherein R′, R^(2′), R^(3′), and K²⁷-K³⁶ are asdisclosed herein. In further embodiments, each of K²⁷-K³⁶ of formula(VIb) is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In still further embodiments, each of K²⁷-K³⁶is H.

In further embodiments, N, R^(2′) and R^(3′) of formula (VIb) togetherform a 5- or 6-membered saturated heterocycle containing at least anitrogen. In still further embodiments, the 5- or 6-membered saturatedheterocycle is substituted or unsubstituted piperidine, morpholine,pyrrolidine, oxazolidine, thiomorpholine, thiazolidine or piperazine. Instill further embodiments, one of R^(2′) and R^(3′) is a Q group asdisclosed herein. In still further embodiments, both R^(2′) and R^(3′)are a Q group and R^(2′) and R^(3′) may be the same or different.

In some embodiments, R^(2′) together with K²⁷ or R^(3′) together withK³⁶ form a part of a 5- or 6-membered saturated or unsaturated ringwherein the ring is optionally substituted. In other embodiments,R^(2′), R^(3′), K²⁷ and K³⁶ together form a part of a saturated orunsaturated bicyclic ring wherein the bicyclic ring is substituted orunsubstituted.

In certain embodiments, K²⁸ and K²⁹ together, or K³⁰ and K³¹ together,or K³³ and K³⁴ together, or K³⁵ and K³⁶ together form a part of a 5- or6-membered saturated or unsaturated ring such as a benzo ring whereinthe 5- or 6-membered saturated or unsaturated ring is substituted orunsubstituted. In further embodiments, K²⁸ and K²⁹ together, or K³⁰ andK³¹ together, or K³³ and K³⁴ together, or K³⁵ and K³⁶ together form abenzo ring wherein the benzo ring is substituted or unsubstituted.

In some embodiments, L has formula (XX):

or a tautomer thereof wherein R^(a), Y³ and K³⁷-K⁴⁴ are as disclosedherein. In other embodiments, Y³ is O or N⁺R^(2′)R^(3′)

In certain embodiments, L has formula (XX) wherein Y³ is O. In otherembodiments, L has formula (XXa):

or a tautomer thereof wherein R^(a), K³⁷-K⁴⁴ are as disclosed herein. Infurther embodiments, each of K³⁷-K⁴⁴ of formula (XXa) is independentlyH, halo, alkyl, halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aryl,aralkyl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino,alkylarylamino, diarylamino, acylamino, hydroxy, thio, thioalkyl,alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano,nitro, sulfhydryl, carbamoyl, trifluoromethyl, phenoxy, benzyloxy,sulfonyl, phosphonyl, sulfonate ester or phosphate ester. In stillfurther embodiments, each of K³⁷-K⁴⁴ is H. In still further embodiments,K³⁷ is Cl or F; and each of K³⁸-K⁴⁴ is H.

In certain embodiments, L has formula (XX) wherein Y³ is N⁺R^(2′)R^(3′).In other embodiments, L has formula (XXb):

or a tautomer thereof wherein R^(a), R^(2′), R^(3′), and K³⁷-K⁴⁴ are asdisclosed herein. In further embodiments, each of K³⁷-K⁴⁴ of formula(XXb) is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In still further embodiments, each of K³⁷-K⁴⁴is H.

In further embodiments, N, R^(2′) and R^(3′) of formula (XXb) togetherform a 5- or 6-membered saturated heterocycle containing at least anitrogen. In still further embodiments, the 5- or 6-membered saturatedheterocycle is substituted or unsubstituted piperidine, morpholine,pyrrolidine, oxazolidine, thiomorpholine, thiazolidine or piperazine. Instill further embodiments, one of R^(2′) and R^(3′) is a Q group asdisclosed herein. In still further embodiments, both R^(2′) and R^(3′)are a Q group and R^(2′) and R^(3′) may be the same or different.

In some embodiments, R^(2′) together with K³⁷ or R^(3′) together withK⁴⁴ form a part of a 5- or 6-membered saturated or unsaturated ringwherein the ring is optionally substituted. In other embodiments,R^(2′), R^(3′), K³⁷ and K⁴⁴ together form a part of a saturated orunsaturated bicyclic ring wherein the bicyclic ring is substituted orunsubstituted.

In certain embodiments, K³⁷ and K³⁸ together, or K³⁹ and K⁴⁰ together,or K⁴² and K⁴³ together form a part of a 5- or 6-membered saturated orunsaturated ring such as a benzo ring wherein the 5- or 6-memberedsaturated or unsaturated ring is substituted or unsubstituted. Infurther embodiments, K³⁷ and K³⁸ together, or K³⁹ and K⁴⁰ together, orK⁴² and K⁴³ together form a benzo ring wherein the benzo ring issubstituted or unsubstituted.

In some embodiments, L has formula (XXI):

or a tautomer thereof wherein R^(b), Y⁴ and K⁴⁵-K⁵² are as disclosedherein. In other embodiments, Y⁴ is O or N⁺R^(2′)R^(3′)

In certain embodiments, L has formula (XXI) wherein Y⁴ is O. In otherembodiments, L has formula (XXIa):

or a tautomer thereof wherein R^(b), K⁴⁵-K⁵² are as disclosed herein. Infurther embodiments, each of K⁴⁵-K⁵² of formula (XXIa) is independentlyH, halo, alkyl, halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aryl,aralkyl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino,alkylarylamino, diarylamino, acylamino, hydroxy, thio, thioalkyl,alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano,nitro, sulfhydryl, carbamoyl, trifluoromethyl, phenoxy, benzyloxy,sulfonyl, phosphonyl, sulfonate ester or phosphate ester. In stillfurther embodiments, each of K⁴⁶-K⁵¹ is H, and at least one of K⁴⁵ andK⁵² is independently Cl or F.

In certain embodiments, L has formula (XXI) wherein Y⁴ isN⁺R^(2′)R^(3′). In other embodiments, L has formula (XXIb):

or a tautomer thereof wherein R^(b), R^(2′), R^(3′), and K⁴⁵-K⁵² are asdisclosed herein. In further embodiments, each of K⁴⁵-K⁵² of formula(XXIb) is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thio, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester or phosphate ester. In still further embodiments, each of K⁴⁵-K⁵²is H.

In further embodiments, N, R^(2′) and R^(3′) of formula (XXIb) togetherform a 5- or 6-membered saturated heterocycle containing at least anitrogen. In still further embodiments, the 5- or 6-membered saturatedheterocycle is substituted or unsubstituted piperidine, morpholine,pyrrolidine, oxazolidine, thiomorpholine, thiazolidine or piperazine. Instill further embodiments, one of R^(2′) and R^(3′) is a Q group asdisclosed herein. In still further embodiments, both R^(2′) and R^(3′)are a Q group and R^(2′) and R^(3′) may be the same or different.

In some embodiments, R^(2′) together with K⁴⁵ or R^(3′) together withK⁵² form a part of a 5- or 6-membered saturated or unsaturated ringwherein the ring is optionally substituted. In other embodiments,R^(2′), R^(3′), K⁴⁵ and K⁵² together form a part of a saturated orunsaturated bicyclic ring wherein the bicyclic ring is substituted orunsubstituted.

In certain embodiments, K⁴⁶ and K⁴⁷ together, or K⁴⁸ and K⁴⁹ together,or K⁵¹ and K⁵² together form a part of a 5- or 6-membered saturated orunsaturated ring such as a benzo ring wherein the 5- or 6-memberedsaturated or unsaturated ring is substituted or unsubstituted. Infurther embodiments, K⁴⁶ and K⁴⁷ together, or K⁴⁸ and K⁴⁹ together, orK⁵¹ and K⁵² form a benzo ring wherein the benzo ring is substituted orunsubstituted.

In some embodiments, each of R, R′, R″, R^(a) and R^(b) is independentlya substituted or unsubstituted phenyl having formula (VII):

-   -   wherein each of R^(4′), R^(5′), R^(6′), R^(7′) and R^(8′) is        independently H, alkyl, alkenyl, alkynyl, heteroalkyl,        cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylaryl,        arylalkyl, heterocyclyl, hydroxy, alkoxy, alkoxyalkyl,        alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogentaed        alkylcarbonylalkyl such as trifluoromethylcarbonylalkyl,        aminoalkyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl,        aminocarbonyl, or NR⁹R¹⁰ or R^(4′) and R^(5′) together, R^(5′)        and R^(6′) together, R^(6′) and R^(7′) together or R^(7′) and        R^(8′) together forming a part of a 5- or 6-membered cycloalkyl,        heterocyclyl, aryl or heteroaryl ring fused with the phenyl ring        of formula (VII); and    -   each of R⁹ and R¹⁰ is independently H, alkyl, alkenyl, alkynyl,        alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl,        or polyether.

In certain embodiments, R^(4′), R^(5′), R^(6′) and R^(7′) isindependently H; and R^(8′) is —COOH, —COR¹⁷, —COOR¹⁸, or —CONR¹⁹R²⁰,wherein R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,alkylaryl, arylalkyl, heterocyclyl, hydroxy, alkoxy, alkoxyalkyl,alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogentaed alkylcarbonylalkyl,aminoalkyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl,aminocarbonyl, or NR⁹R¹⁰, or N, R¹⁹ and R²⁰ together forming a 5- or6-membered heterocycle having at least a nitrogen atom. In otherembodiments, R^(8′) is —CONR¹⁹R²⁰ and N, R¹⁹ and R²⁰ together form a 5-or 6-saturated heterocycle. In further embodiments, the heterocycle issubstituted or unsubstituted piperidine, morpholine, pyrrolidine,oxazolidine, thiomorpholine, thiazolidine or piperazine. In stillfurther embodiments, R^(8′) is a —COOH group. In still furtherembodiments, each of R^(4′), R^(5′), R^(6′) and R^(7′) is H; and R^(8′)is a —COOH group. In some embodiments, each of R^(4′), R^(5′), R^(6′)and R^(7′) is independently H; and R^(8′) is methyl, methoxy or anyother group that can provide sufficient steric hinderance to cause thebenzene ring out of the plane of the polycyclic ring such as thexanthenes ring.

In some embodiments, L has one of formulae (a)-(v):

or a tautomer thereof, wherein each of formulae (a)-(v) is independentlyunsubstituted or substituted.

In certain embodiments, Q of formula (I) is a substituted orunsubstituted phenyl having formula (VIIa):

wherein R⁴, R⁵, R⁶, R⁷ and R⁸ is independently H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,alkylaryl, arylalkyl, heterocyclyl, hydroxy, alkoxy, alkoxyalkyl,alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogentaed alkylcarbonylalkylsuch as trifluoromethylcarbonylalkyl, aminoalkyl, carboxyalkyl,alkoxycarbonyl, alkoxycarbonylalkyl, aminocarbonyl, or NR⁹R¹⁰, or R⁴ andR⁵ together, R⁵ and R⁶ together, R⁶ and R⁷ together or R⁷ and R⁸together forming a part of a 5- or 6-membered cycloalkyl, heterocyclyl,aryl or heteroaryl ring fused with the phenyl ring of formula (VIIa);and

-   -   each of R⁹ and R¹⁰ is independently H, alkyl, alkenyl, alkynyl,        alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl,        or polyether.

In certain embodiments, R⁶ of formula (VIIa) is NR⁹R¹⁰. In otherembodiments, R¹ of formula (I) is H, alkyl, halogenated alkyl,heteroalkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl,cycloalkyl, cycloalkenyl, and cycloalkynyl; each of R⁴ R⁵, R⁶, R⁷ and R⁸of formula (VIa) is independently H, halogen, alkyl, alkoxy, orpolyether; R⁶ is OR¹¹ or CH₂CH₂COR , where R¹¹ is H, alkyl, alkoxyalkyl,alkanoyl, or polyether; R¹² is an electron-withdrawing group selectedfrom CF₃, halogen-substituted lower alkyl (e.g., CF_(n)H_(3-n), whereinn is 1, 2, or 3), or (C═O)—O—V², wherein V² is a group selected fromalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,alkaryl or arylalkyl.

In some embodiments, R⁶ of Q is —OCH₂OCH₃, OH, NR⁹R¹⁰, —CH₂CH₂C(═O)CF₃,or —CH₂CH₂C(═O)OCH₃ where each of R⁹ and R¹⁰ is independently H oralkyl; and each of R⁴, R⁵, R⁷ and R⁸ is H. In other embodiments, R⁶ isOH, NH₂ or —CH₂CH₂C(═O)CF₃.

In certain embodiments, R¹ is H, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, and cycloalkynyl; each of R⁴, R⁵, R⁶, R⁷ and R⁸ isindependently H, halogen, alkyl, alkoxy, or polyether; R⁶ is OR¹¹ orCH₂CH₂COR¹², where R¹¹ is H, alkyl, alkoxyalkyl, alkanoyl, or polyether;R¹² is an electron-withdrawing group selected from CF₃,halogen-substituted lower alkyl, or (C═O)—O—V², wherein V² is a groupselected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, alkaryl or arylalkyl.

In some embodiments, when L has formula (II) where Y is NR²R³, then R⁶of Q is hydroxy, alkenyl, alkynyl, heteroalkyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclyl, alkoxyalkyl,alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogentaed alkylcarbonylalkylsuch as trifluoromethylcarbonylalkyl, carboxyalkyl, alkoxycarbonyl,alkoxycarbonylalkyl or aminocarbonyl, or R⁴ and R⁵ together, R⁵ and R⁶together, R⁶ and R⁷ together or R⁷ and R⁸ together form a 5- or6-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ring fused withthe phenyl ring of formula (VIIa).

In some embodiments, the aromatic amine compounds have formula (I):

wherein L has one of formulae (II)-(VI), (XX), (XXI), (Va)-(Vd),(VIa)-(VIb), (XXa)-(XXb), (XXIa)-(XXIb), and (a)-(v) as disclosedherein; Q has formula (VIIa) as disclosed herein; and R¹ is as disclosedherein.

When any of the aromatic amine compounds, such as formula (IVa), formula(Vb), formula (VIIb), formula (IXb) or Compound 3, is positivelycharged, the positive charge may be balanced by any suitablecounteranion known to a skilled artisan. Some non-limiting examples ofsuitable counteranion include halides such as fluoride, chloride,bromide and iodide, carboxylates such as formate and acetate, hydrogencarbonate, nitrate, nitrite and the like. In some embodiments, thecounteranion is chloride. In some embodiments, the counteranion isacetate.

In certain embodiments, the aromatic amine compounds disclosed hereininclude Compounds 1a-1d, 2, 3, 4a-4e and 5a-5d:

or a tautomer thereof, wherein each of Compounds 1a-1d, 2, 3, 4a-4e,5a-5d, 10, 12, 12a, 14, 22, and 30 is independently substituted orunsubstituted.

In certain embodiments, the aromatic amine compound is Compound (10). Inother embodiments, the aromatic amine compound is Compound (12). Infurther embodiments, the aromatic amine compound is Compound (12a). Instill further embodiments, the aromatic amine compound is Compound (14).In still further embodiments, the aromatic amine compound is Compound(22). In other embodiments, the aromatic amine compound is Compound(30).

In some embodiments, the aromatic amine compound is Compound (30). Inother embodiments, the tautomer of Compound (30) has formlua (30a) asshown below.

Aromatic Amine Compounds as Luminescence Quenchers

In certain embodiments, the aromatic amine compounds having formula (I)can be used as luminescence quenchers or luminescence quenchingcompounds. In certain embodiments, the luminescence quenching compoundshave formula (VIII) or (IX):

wherein Y¹, Y², Q, R′, R¹ and K²¹-K³⁶ are as disclosed herein.

In certain embodiments, each of Y¹ or Y² is independently O. In otherembodiments, each of Y¹ or Y² is independently N⁺R^(2′)R^(3′). Infurther embodiments, each of Y¹ or Y² is independently NR^(2′)R^(3′). Instill further embodiments, the luminescence quenching compounds haveformula (VIIIa), (VIIIb), (IXa) or (IXb):

wherein Q, R′, R¹ and K²¹-K³⁶ are as disclosed herein.

In certain embodiments, N, R^(2′) and R^(3′) of formula (VIIb) or (IXb)together form a 4-, 5-, 6-, 7- or 8-membered saturated heterocyclecontaining at least a nitrogen. In other embodiments, N, R^(2′) andR^(3′) of formula (VIIb) or (IXb) together form a 5- or 6-memberedsaturated heterocycle. In further embodiments, N, R^(2′) and R^(3′) offormula (VIIb) or (IXb) together form a 5- or 6-membered saturatedheterocycle selected from substituted or unsubstituted piperidine,morpholine, pyrrolidine, oxazolidine, thiomorpholine, thiazolidine orpiperazine. In still further embodiments, one of R^(2′) and R^(3′) offormula (VIIb) or (IXb) is Q as disclosed herein. In still furtherembodiments, both R^(2′) and R^(3′) are Q and R^(2′) and R^(3′) may bethe same or different.

In some embodiments, N, Q and R¹ of formula (VIII), (VIII), (VIIIa),(VIIIb), (IXa) or (IXb) together form a 5- or 6-membered saturatedheterocycle containing at least a nitrogen. In other embodiments, the 5-or 6-membered saturated heterocycle is substituted or unsubstitutedpiperidine, morpholine, pyrrolidine, oxazolidine, thiomorpholine,thiazolidine or piperazine. In further embodiments, R¹ is a Q group asdisclosed herein. In still further embodiments, both R¹ and Q group maybe the same or different.

In certain embodiments, R^(2′) together with K²¹ or R^(3′) together withK²² of formula (VIIIb) form a part of a 5- or 6-membered saturated orunsaturated ring wherein the ring is optionally substituted. In otherembodiments, R^(2′), R^(3′), K²¹ and K²² together form a part of asaturated or unsaturated bicyclic ring wherein the bicyclic ring issubstituted or unsubstituted.

In some embodiments, R^(2′) together with K²⁷ or R^(3′) together withK³⁶ of formula (IXb) form a part of a 5- or 6-membered saturated orunsaturated ring wherein the ring is optionally substituted. In otherembodiments, R^(2′), R^(3′), K²⁷ and K³⁶ together form a part of asaturated or unsaturated bicyclic ring wherein the bicyclic ring issubstituted or unsubstituted.

In certain embodiments, K²² and K²³ together or K²⁵ and K²⁶ togetherform a part of a 5- or 6-membered saturated or unsaturated ring such asa benzo ring wherein the 5- or 6-membered saturated or unsaturated ringis substituted or unsubstituted. In further embodiments, K²² and K²³together or K²⁵ and K²⁶ together form a benzo ring wherein the benzoring is substituted or unsubstituted.

In some embodiments, the luminescence quenching compounds includeCompounds 4a-4e and 5a-5d:

wherein each of Compounds 4a-4e and 5a-5d is independently substitutedor unsubstituted.

In some embodiments, at least one of the L, R¹ and Q groups of thearomatic amine compounds of formula (I) is substituted by a reactivegroup (Rg) or a conjugated group (Cg) wherein Rg or Cg is optionallyattached to the aromatic amine compounds disclosed herein through alinkage group, —X—. In other embodiments, at least one of the L, R¹ andQ groups of the aromatic amine compounds disclosed herein is substitutedby an —X-Rg or —X-Cg group. In other embodiments, the L group of thearomatic amine compounds of formula (I) is substituted by an —X-Rg or—X-Cg group.

In some embodiments, X is or comprises a bond or a linking group such asO, S, an aminylene group (e.g., an NR group where R is H, an alkylgroup, an alkenyl group, an alkynyl group, a carboxyl group, an acylgroup, an aromatic group, or a heterocyclic group), a sulfonyl group, anorganic linking group, or a combination thereof. The organic linkinggroup disclosed herein may be a divalent linking organic groupconnecting any of two fragments, such as L, R¹, Q, Rg or Cg, of achemical formula together. Some non-limiting examples of the divalentorganic linking group include a carbonyl group, an alkylene group, anarylene group, a divalent heterocyclic group, and combinations thereof.Another non-limiting example of the divalent organic linking groupincludes a —(CH₂)_(m)— group, where m is an integer between 1 and 50,inclusive, and one or more of the methylene groups is optionallyreplaced by O, S, N, C, B, Si, P, C═O, O═S═O, a heterocyclic group, anaromatic group, an NR_(a) group, a CR_(b) group, a CR_(c)R_(d) group, aSiR_(e)R_(f) group, a BR_(g) group, or a P(═O)R_(h) group, where R_(a),R_(b), R_(c), R_(d), R_(e), R_(f), R_(g), and R_(h) are, eachindependently, a bond, H, a hydroxyl group, a thiol group, a carboxylgroup, an amino group, a halogen, an acyl group, an alkoxy group, analkylsulfanyl group, an alkenyl group, such as a vinyl group, an allylgroup, and a 2-phenylethenyl group, an alkynyl group, a heterocyclicgroup, an aromatic group, a part of a ring group, such as cycloalkylgroups, heterocyclic groups, and a benzo group, or an alkyl group whereone or more of the hydrogens of the alkyl group is optionally replacedby an aromatic group, a hydroxyl group, a thiol group, a carboxyl group,an amino group, or a halogen. A non-limiting example of the aminylenegroup includes an NR group where R is H, an alkyl group, an alkenylgroup, an alkynyl group, an acyl group, an aromatic group, and aheterocyclic group.

In certain embodiments, the organic linking group may have a valence of3 or more and, therefore, may link any of 3 or more fragments, such asL, R¹, Q, Rg or Cg, of a chemical formula together. A non-limitingexample of an organic linking group having a valence of 3 is a trivalentorganic linking group created by replacing a methylene group in the—(CH₂)_(m)— group with a CR_(b) group. Another non-limiting example ofan organic linking group having a valence of 4 is a tetravalent organiclinking group created by replacing a methylene group in the —(CH₂)_(m)—group with a carbon atom. Another non-limiting example of an organiclinking group having a valence of 3 is a trivalent organic linking groupcreated by replacing a methylene group in the —(CH₂)_(m)— group with N,P, or B. A further non-limiting example of an organic linking grouphaving a valence of 4 is a tetravalent organic linking group created byreplacing two methylene groups in the —(CH₂)_(m)— group with two CR_(b)groups. Based on the disclosure herein, a person skill in the art maycreate an organic linking group having a valence greater than 2 byreplacing at least one methylene group in the —(CH₂)_(m)— group with atleast an atom or a group having a valence of 3 or more, such as N, P, B,C, Si, a CR_(b) group, an aromatic group having a valence greater than2, and a heterocyclic group having a valence greater than 2.

In other embodiments of interest, the organic linking group may compriseat least an unsaturated bond, such as a —CR_(b)═N— bond, a double bondor a triple bond. A non-limiting example of an organic linking grouphaving a double bond is an unsaturated organic linking group created byreplacing two adjacent methylene groups in the —(CH₂)_(m)— group withtwo CR_(b) groups. The double bond is located between the two adjacentCR_(b) groups. Another non-limiting example of an organic linking grouphaving a triple bond is an unsaturated organic linking group created byreplacing two adjacent methylene groups in the —(CH₂)_(m)— group withtwo carbon atoms respectively. The triple bond is located between thetwo adjacent carbon atoms. Another non-limiting example of an organiclinking group having a —CR_(b)═N— bond is an unsaturated organic linkinggroup created by replacing two adjacent methylene groups in the—(CH₂)_(m)— group with one CR_(b) group and an N atom. Based on thedisclosure herein, a person skill in the art may create an organiclinking group having at least an unsaturated bond by replacing at leastone pair of adjacent methylene groups in the —(CH₂)_(m)— group, eachindependently, with an atom or a group selected from the groupconsisting of N, P, B, C, Si, a CR_(b) group, an aromatic group having avalence greater than 2, and a heterocyclic group having a valencegreater than 2.

In certain embodiments, one or more of R′, R^(2′), R^(3′), R¹-R¹², andK²¹-K³⁶ are independently substituted by -Cv-Rg or -Cv-Cg groups. Inother embodiments, one or more of Q, R′, R^(2′), R^(3′) and R¹¹-R¹² areindependently substituted by -Cv-Rg or -Cv-Cg groups.

The luminescence quenching compounds having a reactive group (Rg) maycomprise a wide variety of organic or inorganic substances that containor are modified to contain at least one functional group with suitablereactivity toward the Rg group which result in chemical attachment ofthe reactive group (Rg), represented by -Cv-Rg. In some embodiments, thereactive group (Rg) and functional group are respectively anelectrophile and a nucleophile that can react to generate a covalentlinkage. The conjugation reaction between the reactive group (Rg) andfunctional group at the conjugated substance (Cg) results in one or moreatoms of the reactive group (Rg) to be incorporated into the linkage,Cv, which attaches the compound with reactive group (Rg) to theconjugated substance (Cg). Some non-limiting examples of the reactivegroup (Rg) and the respective functional group are listed in Table 1.The tabulation is not meant to be inclusive of chemical reactivity sincewith the appropriate choice of solvent, co-solvent, stoichiometricratio, temperature, pressure, reaction time, pH, catalyst and the like,other functional groups can be made to react with the reactive sitesdisclosed herein whereas the functional groups disclosed herein can bemade to react with other reactive sites. Some non-limiting examples ofsuitable reactive groups (Rg) include acrylamide, acyl azide, acylhalide, nitrile, aldehyde, ketone, alkyl halide, alkyl sulfonate,anhydride, aryl halide, alkyne, alcohol, amine, carboxylic acid,carbodiimide, diazoalkane, epoxide, haloacetamide, hydroxylamine,hydrazine, imido ester, isothiocyanate, maleimide, sulfonate ester orsulfonyl halide.

TABLE 1 Functional Group Reactive group (Electrophile) (Nucleophile)Resulting Linkage activated esters amines/anilines amides (succinimidylesters) acrylamides thiols thioethers acyl azides amines/anilines amidesacyl halides amines/anilines amides acyl halides Alcohols/phenols estersacyl nitriles alcohols/phenols esters acyl nitriles amines/anilinesamides aldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines amides aryl halides thiols thiophenolsaryl halides amines aryl amines alkynes azides triazoles alcohols acidderivatives esters amines carboxylic acids amides amines halides alkylamines amines aldehydes/ketones imines carboxylic acids amines/anilinesamides carboxylic acids alcohols esters carboxylic acids hydrazineshydrazides carbodiimides carboxylic acids N-acylureas or anhydridesdiazoalkanes carboxylic acids esters epoxides thiols thioestershaloacetamides thiols thioethers hydroxylamines aldehydes/ketones oximeshydrazines aldehydes/ketones hydrazones imido esters amines/anilinesamidines isothiocyanates amines/anilines thioureas isothiocyanatesalcohols/phenols isourethanes maleimides thiols thioethers maleimidesamines amines sulfonate esters amines/anilines alkyl amines sulfonateesters thiols thioesters sulfonate esters carboxylic acids esterssulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate esters

The reactive group in the luminescence quenching compounds disclosedherein is useful for the preparation of any conjugated substance thatbears a suitable functional group for covalent linkage of the two. Somenon-limiting examples of suitable conjugates include conjugates ofantigens, steroids, vitamins, drugs, haptens, metabolites, toxins, aminoacids, peptides, nucleotides, oligonucleotides, nucleic acid,carbohydrates, lipids, and so on. Choice of the reactive group used toattach the luminescence quenching compounds disclosed herein to thesubstance to be conjugated typically depends on the functional group onthe substance to be conjugated and the type or length of covalentlinkage desired. The types of functional groups typically present on thesubstances include, but are not limited to, amines, thiols, alcohols,phenols, aldehydes, ketones, phosphates, imidazoles, hydrazines,hydroxylamines or a combination of these groups.

In some embodiments, the conjugated substance is additionally conjugatedto one or more luminophores, which may be the same or different. Inother embodiments, energy transfer from the luminophore to the quenchingcompound occurs, resulting in significant quenching of luminescence.

The applications of luminescence quenching compounds arewell-documented, simply as calorimetric labels for a conjugatedsubstance, or in Fluorescence Resonance Energy Transfer (FRET)technology. Some non-limiting examples of such applications aredescribed in U.S. Pat. No. 6,399,392; and The Handbook: a Guide toFluorescent Probes and Labeling Technologies, 10th Edition, MolecularProbes, 2006, both of which are incorporated herein by reference.

Aromatic Amine Compounds as Fluorogenic Probes for ReactiveOxygen/Nitrogen Species

In certain embodiments, the aromatic amine compounds having formula (I)can be used as fluorogenic probe compounds or fluorogenic probecompositions. The fluorogenic probe compounds can be used as fluorogenicprobes for reactive oxygen species (ROS) or reactive nitrogen species(RNS) such as ¹O₂, O₂ ^(•−), NO, H₂O₂, .OH, ⁻OCl, ONOO⁻ and alkylperoxylradical (ROO^(•)). In some embodiments, Q of formula (I) is asubstituted phenyl, which can be cleaved oxidatively by certain ROS orRNS to release of the corresponding L-NHR¹ luminophore or fluorophorehaving strong luminescence or fluorescence properties.

In some embodiments, the fluorogenic probe compounds have formula (X):

wherein L, R¹ and R⁴-R⁸ are as disclosed herein. In some embodiments, Lis a fluorophore. In other embodiments, L has one of formulae (II)-(VI),(XX), (XXI), (Va)-(Vd), (VIa)-(VIb), (XXa)-(XXb), (XXIa)-(XXIb), and(a)-(v) disclosed herein.

In certain embodiments, R¹ and R⁴ together or R¹ and R⁸ together form apart of a 5- or 6- or 7-membered cycloalkyl, heterocyclyl, aryl orheteroaryl ring which is fused with the phenyl ring of formula (X).

In some embodiments, each of R⁴-R⁸ is H. In some embodiments, each ofR⁴, R⁵, R⁷ and R⁸ is H; and R⁶ is H, hydroxy, methoxyl,trifluoromethylcarbonylethyl, methyoxycarbonylalkyl or methoxymethoxy.

In general, the fluorogenic probe having formula (X) disclosed hereincan react with reactive oxygen species and/or reactive nitrogen speciesto form tetra-substituted ammonium (XI), which undergo hydrolysis togenerate L-derived fluorophore (XII) and a quinone-type by-product(XIII). This general reaction is shown in Scheme 1 below where L, R¹ andR⁴-R⁸ are as disclosed herein and where R^(6″) is derived from R⁶ byremoving a hydrogen or a monovalent group such as alkyl from R⁶.

In some embodiments, the fluorogenic probe having formula (X) disclosedherein can react with only one or two or three kinds of reactive oxygenspecies or reactive nitrogen species to generate fluorophores havingformula (X) in substantially higher yields than that of the others. Incertain embodiments, the fluorogenic probe having formula (X) can reactwith peroxynitrite, hypochlorite or hydroxy radical in a higher yieldthan that of any other ROS and RNS. In other embodiments, thefluorogenic probe having formula (X) reacts with peroxynitrite in ahigher yield than that of any other ROS and RNS. In further embodiments,the fluorogenic probe having formula (X) reacts with hypochlorite inhigher yields than that of any other ROS and RNS. In still furtherembodiments, the fluorogenic probe having formula (X) reacts withhydroxy radical in a higher yield than that of any other ROS and RNS.

In certain embodiments, the fluorogenic probe having formula (X) reactswith peroxynitrite, hypochlorite or hydroxy radical in a yield about 5%higher than, about 10% higher than, about 15% higher than, about 20%higher than, about 25% higher than, about 30% higher than, about 35%higher than, about 40% higher than, about 45% higher than, about 50%higher than, about 50% higher than, about 60% higher than, about 65%higher than, about 70% higher than, about 75% higher than, about 80%higher than, about 85% higher than, about 90% higher than or about 95%higher than that of any other ROS and RNS.

The fluorogenic probes provided herein can be used to detectperoxynitrite specifically. In some embodiments, at least one of R⁴, R⁵,R⁶, R⁷ and R⁸ of formula (X) of formula (X) is —CH₂CH₂C(═W)R¹³, whereinR¹³ is an electron-withdrawing group selected from CF₃,halogen-substituted lower alkyl (e.g., CF_(n)H_(3-n), where n is 1, 2,or 3), —O—V¹, or (C═O)—O—V², wherein V¹ and V² are groups selected fromalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,alkaryl or arylalkyl.

In certain embodiments, the fluorogenic probes disclosed herein haveformula (XIVa), (XIVb) or (XIVc):

-   -   wherein L, R¹, R⁴, R⁵, R⁷, R⁸ are defined herein;    -   W is O or S; and    -   R¹³ is an electron-withdrawing group selected from CF₃,        halogen-substituted lower alkyl (e.g., CF_(n)H_(3-n), wherein n        is 1, 2, or 3), —O—V¹, or (C═O)—O—V², wherein V¹ and V² are        groups selected from alkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl.

In some embodiments, W is O. In other embodiments, R¹³ is CF₃. Infurther embodiments, R¹ is methyl. In further embodiments, W is O; R¹³is CF₃; each of R⁴, R⁵, R⁷ and R⁸ is H; and R¹ is methyl.

In certain embodiment, the fluorogenic probes having formula (XIV)reacts with peroxynitrite specifically to form a dioxirane intermediatewhich subsequently oxidizes the phenyl ring of formula (XIV) to causethe C—N bond cleavage and therefore the release of L derivative (XII) asshown in Scheme 2 below. The L derivative (XII) can emit a strongfluorescence signal when excited.

The fluorogenic probes disclosed herein can be used for detectingperoxynitrite with high sensitivity. In some embodiments, at least oneof R⁴, R⁵, R⁶, R⁷ and R⁸ of the fluorogenic probes of formula (X) isOR¹⁴ where R¹⁴ is H, alkyl, alkoxyalkyl, alkanoyl or polyether. In otherembodiments, the fluorogenic probes provided herein have formula (XVIa),(XVIb) or (XVIc):

-   -   wherein L, R¹, R⁴, R⁵, R⁶, R⁷ and R⁸ are as disclosed herein;        and    -   R¹⁴ is H, alkyl, alkoxyalkyl, alkanoyl or polyether.

In some embodiments, R⁵ and R¹⁴ together or R⁷ and R¹⁴ together form a5- or 6- or 7-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ringfused with the phenyl ring of formula (XVIa). In other embodiments, R⁴and R¹⁴ together or R⁶ and R¹⁴ together form a 5- or 6- or 7-memberedcycloalkyl, heterocyclyl, aryl or heteroaryl ring fused with the phenylring of formula (XVIb). In further embodiments, R⁵ and R¹⁴ together forma 5- or 6- or 7-membered cycloalkyl, heterocyclyl, aryl or heteroarylring fused with the phenyl ring of formula (XVIc).

In certain embodiment, each of R⁴, R⁵, R⁷, R⁸ and R¹⁴ of formula (XVIa)is H; and R¹ is methyl. In other embodiment, each of R⁴, R⁶, R⁷, R⁸ andR¹⁴ of formula (XVIb) is H; and R¹ is methyl. In further embodiment,each of R⁵, R⁶, R⁷, R⁸ and R¹⁴ of formula (XVIc) is H; and R¹ is methyl.

In some embodiment, the fluorogenic probes having formula (XVIa)substantially reacts with peroxynitrite specifically to form dioxiraneintermediate which subsequently oxidizes the phenyl ring of formula(XVIa) to cause the C—N bond cleavage and therefore the release of Lderivative (XII) as shown in Scheme 3 below. The L derivative (XII) canemit a strong fluorescence signal when excited.

The fluorogenic probes disclosed herein can be used for detectinghypochlorite with high sensitivity. In some embodiments, at least one ofR⁴, R⁵, R⁶, R⁷ and R⁸ of the fluorogenic probes of formula (X) isNR¹⁵R¹⁶ where each of R¹⁵ and R¹⁶ is H, alkyl, alkenyl, alkynyl,alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl, or polyether. In otherembodiments, the fluorogenic probes provided herein have formula(XVIIIa), (XVIIIb) or (XVIIIc):

-   -   wherein L, R¹, R⁴, R⁵, R⁶, R⁷ and R⁸ are as disclosed herein;        and    -   each of R¹⁵ and R¹⁶ is independently H, alkyl, alkenyl, alkynyl,        alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl,        cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl or        polyether.

In some embodiments, R⁵ and R¹⁵ together or R⁷ and R¹⁶ together form a5- or 6- or 7-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ringfused with the phenyl ring of formula (XVIIIa). In other embodiments, R⁴and R¹⁵ together or R⁶ and R¹⁶ together form a 5- or 6- or 7-memberedcycloalkyl, heterocyclyl, aryl or heteroaryl ring fused with the phenylring of formula (XVIIIb). In further embodiments, R⁵ and R¹⁵ togetherform a 5- or 6- or 7-membered cycloalkyl, heterocyclyl, aryl orheteroaryl ring fused with the phenyl ring of formula (XVIIIc).

In some embodiment, the fluorogenic probes having formula (XVIIIa)substantially reacts with hypochlorite specifically to cause the C—Nbond cleavage and therefore the release of L derivative (XII) as shownin Scheme 4 below. The L derivative (XII) can emit a strong fluorescencesignal when excited.

In certain embodiment, each of R⁴, R⁵, R⁷ and R⁸ of formula (XVIIIa) isH; R¹ is methyl; and each of R¹⁵ and R¹⁶ is independently H or methyl.In certain embodiment, each of R⁴, R⁶, R⁷ and R⁸ of formula (XVIIIb) isH; R¹ is methyl; and each of R¹⁵ and R¹⁶ is independently H or methyl.In certain embodiment, each of R⁵, R⁶, R⁷ and R⁸ of formula (XVIIIc) isH; R¹ is methyl; and each of R¹⁵ and R¹⁶ is independently H or methyl.

The fluorogenic probes for reactive oxygen species and/or reactivenitrogen species disclosed herein comprise rhodol, rhodamine, resorufin,and naphthofluorescein fluorophores. In some embodiments, the compoundsor peroxynitrite probes or hypochlorite probes disclosed herein aresubstantially non-fluorescent. In other embodiments, the compounds orperoxynitrite probes or hypochlorite probes disclosed herein canefficiently react with peroxynitrite or hypochlorite under physiologicalconditions to give a strong fluorescent signal. In further embodiments,the amount of peroxynitrite or hypochlorite can be determined with veryhigh specificity and selectivity by measuring the fluorescent signal ofthe oxidized probes.

In some embodiment, L of the fluorogenic probes of formula (I) can beany fluorophore known to a skilled artisan. In other embodiments, L isderived from rhodol, rhodamine, resorufin, naphthofluorescein,seminaphthofluorescein or a derivative thereof.

In some embodiments, L is derived from a rhodol, rhodamine or derivativethereof, wherein L has formula (Vd):

wherein Y¹ and K²¹-K²⁶ are as disclosed herein.

In some embodiments, R″ of formula (Vd) is a substituted orunsubstituted phenyl having formula (VII):

wherein R^(4′), R^(5′), R^(6′), R^(7′) and R^(8′) are as disclosedherein. In certain embodiments, one or more of R^(4′), R^(5′), R^(6′),R^(7′) and R^(8′) of formula (VII) is halogenated alkyl. In otherembodiments, one or more of R^(4′), R^(5′), R^(6′), R^(7′) and R^(8′) offormula (VII) is chloromethyl, which can react with sulfide groups incells to keep the fluorogenic probes inside the cells and from leakage.In other embodiments, at least one of R^(4′), R^(5′), R^(6′), R^(7′) andR^(8′) of formula (VII) is linked with a cell organelle localizationmoiety such as triphenylphosphonium. In further embodiments, each ofR^(4′), R^(5′), R^(6′) and R^(7′) of formula (VII) is H; and R^(8′) is a—COOH group, methyl, or methoxy.

In certain embodiments, L is derived from a resorufin or a derivativethereof and has formula (Va):

wherein Y¹ and K²¹-K²⁶ are as disclosed herein. In some embodiments, Y¹is O. In other embodiments, Y¹ is N⁺R^(2′)R^(3′). In furtherembodiments, each of K²¹-K²⁶ of formula (Va) is independently H, halo,alkyl, halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl,alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino,alkylarylamino, diarylamino, acylamino, hydroxy, thio, thioalkyl,alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, cyano,nitro, sulfhydryl, carbamoyl, trifluoromethyl, phenoxy, benzyloxy,sulfonyl, phosphonyl, sulfonate ester or phosphate ester. In otherembodiments, each of K²¹-K²⁶ is H. In other embodiments, each of K²² andK²⁵ is independently chlorine or fluorine.

In some embodiments, L is derived from a naphthofluorescein or aderivative thereof and has formula (VI):

wherein Y² and K²⁷-K³⁶ are as disclosed herein. In other embodiments, Y²is N⁺R^(2′)R^(3′) wherein R^(2′) and R^(3′) are as disclosed herein. Infurther embodiments, Y² is O.

In some embodiments, R′ is a substituted or unsubstituted phenyl havingformula (VII):

wherein R^(4′), R^(5′), R^(6′), R^(7′) and R^(8′) are as disclosedherein. In some embodiments, R^(4′) or R^(8′) of formula (VII) is a—COOH group. In further embodiments, each of R^(4′), R^(5′), R^(6′) andR^(7′) of formula (VII) is H; and R^(8′) is a —COOH group, methyl ormethoxy.

In some embodiments, L is derived from a seminaphthofluorescein or aderivative thereof and has formula (XX) or (XXI):

wherein Y³, Y⁴ and K³⁷-K⁵² are as disclosed herein. In otherembodiments, each of Y³ and Y⁴ is independently N⁺R^(2′)R^(3′) whereinR^(2′) and R^(3′) are as disclosed herein. In further embodiments, eachof Y³ and Y⁴ is O. In some embodiments, K³⁷ is chlorine or fluorine. Insome embodiments, at least one of K⁴⁵ and K⁵² is chlorine or fluorine.

In some embodiments, R^(a) or R^(b) is a substituted or unsubstitutedphenyl having formula (VII):

wherein R^(4′), R^(5′), R^(6′), R^(7′) and R^(8′) are as disclosedherein. In some embodiments, R^(4′) or R^(8′) of formula (VII) is a—COOH group. In further embodiments, each of R^(4′), R^(5′), R^(6′) andR^(7′) of formula (VII) is H; and R^(8′) is a —COOH group, methyl ormethoxy.

In certain embodiments, the fluorogenic probe compositions can be usedfor measuring, detecting or screening peroxynitrite, wherein thefluorogenic probe compositions comprise the aromatic amine compounddisclosed herein. In certain embodiments, the aromatic amine compound isCompound (10), Compound (12) or Compound (30):

or a tautomer thereof, or a combination thereof.

In some embodiments, the fluorogenic probe compositions can be used formeasuring, detecting or screening hypochlorite, wherein the fluorogenicprobe compositions comprise the aromatic amine compound disclosedherein. In certain embodiments, the aromatic amine compound is Compound(14):

or a tautomer thereof.

In certain embodiments, the fluorogenic probe compositions disclosedherein further comprise a solvent, an acid, a base, a buffer solution ora combination thereof.

In some embodiments, the aromatic amine compound is Compound (10) whichreacts with ONOO⁻ to form Compound (11) with strong fluorescentproperties as shown in Scheme 5 below. The fluorescence spectra showingfluorescence intensities of Compound 10 in response to differentconcentrations of ONOO⁻ at different wavelengths are shown in FIGS. 1-2.The fluorescence intensities of Compound 10 in response to different ROSand RNS is shown in FIG. 3.

In other embodiments, the aromatic amine compound is Compound (12) whichreacts with ONOO⁻ to form Compound (13) with strong fluorescentproperties as shown in Scheme 6 below. The fluorescence spectra showingfluorescence intensities of Compound 12 in response to differentconcentrations of ONOO⁻ at different wavelengths are shown in FIG. 4.The fluorescence intensities of Compound 12 in response to different ROSand RNS is shown in FIG. 5.

In further embodiments, the aromatic amine compound is Compound (14)which reacts with OCl⁻ to form Compound (13) with strong fluorescentproperties as shown in Scheme 7 below. The fluorescence spectra showingfluorescence intensities of Compound 14 in response to differentconcentrations of OCl⁻ at different wavelengths are shown in FIG. 6. Thefluorescence intensities of Compound 14 in response to different ROS andRNS is shown in FIG. 7.

In some embodiments, the fluorogenic probe compositions for measuring,detecting or screening peroxynitrite comprise Compound (22), Compound(12a) or a combination thereof. Each of Compound (22) and Compound (12a)is an ester derivative of Compound (10) and Compound (12) respectively.In certain embodiments, Compound (22) and Compound (12a) providedesirable cell membrane permeability.

In some embodiments, the fluorogenic probe compositions for measuring,detecting or screening hypochlorous acid/hypochloride comprise Compound(14a), which is an ester derivative of Compound (14). Compound (14a)provides desirable cell membrane permeability.

In certain embodiments, the fluorogenic probe compositions disclosedherein comprise an acetate or acetoxymethyl (AM) ester derivative offluorogenic probe compounds disclosed herein. The neutral forms of thesefluorogenic probe compounds are advantageous for cell membranepermeability.

In certain embodiments, the fluorogenic probe compositions disclosedherein further comprise a solvent, an acid, a base, a buffer solution ora combination thereof.

Also provided herein are reagent compositions for measuring directly orindirectly peroxynitrite or hypochlorite in chemical or biologicalsamples such as microorganism, or a cell or tissue from animals. Thereagent composition comprises the fluorogenic probe disclosed herein. Insome embodiments, the reagent composition further comprises a solvent,an acid, a base, a buffer solution or a combination thereof a base, abuffer solution or a combination thereof.

Also provided herein are methods for measuring peroxynitrite orhypochlorite in a sample. In some embodiments, the methods comprise thesteps of (a) contacting a fluorogenic probe disclosed herein with thesample to form a fluorescent compound; and (b) measuring fluorescenceproperties of the fluorescent compound. In some embodiments, thefluorescence properties are measured with methods disclosed herein orany method known to a person skilled in the art. In other embodiments,the sample is a chemical sample or biological sample. In furtherembodiments, the sample is a biological sample comprising amicroorganism, or a cell or tissue from animals.

Also provided herein are high-throughput screening fluorescent methodsfor detecting peroxynitrite or hypochlorite in samples. In someembodiments, the high-throughput screening fluorescent methods comprisethe steps of (a) contacting a fluorogenic probe disclosed herein withthe samples to form one or more fluorescent compounds; and (b) measuringfluorescence properties of the fluorescent compounds. In otherembodiments, the fluorescence properties are measured with methodsdisclosed herein or any method known to a person skilled in the art.

Also provided herein are high-throughput methods for screening one ormore target compounds that can increase or decrease the level ofperoxynitrite or hypochlorite. In some embodiments, the high-throughputmethods comprise the steps of (a) contacting a fluorogenic probedisclosed herein with the target compounds to form one or morefluorescent compounds; and (b) measuring fluorescence properties of thefluorescent compounds to determine the target compounds quantitativelyor qualitatively. In other embodiments, the fluorescence properties aremeasured with methods disclosed herein or any method known to a personskilled in the art.

In some embodiments, informatics systems can be used and implemented inthe high-throughput methods disclosed herein. In other embodiments, theinformatics systems provide the software control of the physical devicesused in the high-throughput method. In other embodiments, theinformatics systems organize electronic data generated by thehigh-throughput methods. In further embodiments, the informatics systemsstore electronic data generated by the high-throughput methods.

General Synthetic Procedures

The aromatic amine compounds or fluorogenic probes disclosed herein maybe made by one skilled in the art with known organic syntheses as wellas various general or specific synthetic procedures disclosed herein.

Generally, the key steps for the synthesis of the aromatic aminecompounds include a hydroxy activation step, generally using a triflate,and subsequent amination step as shown in Scheme 8 below.

where L, R¹, and Q are as disclosed herein; Tf is triflyl; Pd-cat. is apalladium-ligand catalysis system for C—N bond formation. First, the OHgroup of the luminophore (L-OH) was activated by reacting with atriflyl-donating reagent, such as triflic anhydride, to form a triflategroup. Then the triflate group subsequently underwent cross-couplingreaction with an amine having formula HNR¹Q in the presence of acatalyst, such as a Pd catalyst, to form the aromatic amine compound offormula (I).

Some non-limiting examples of suitable synthetic method can be found inU.S. Patent Application No. 61/041923, filed Apr. 3, 2008, which isincorporated herein by reference.

EXAMPLES

The following Examples 1-13 and FIGS. 1-11 are detailed descriptions ofthe methods of making and using the subject compounds disclosed in thisinvention. The detailed disclosure falls within the scope of, and serveto exemplify, the synthetic schemes or procedures disclosed herein whichform part of this disclosure. These examples, figures and schemes arepresented for illustrative purposes only and are not intended to limitthe scope of this disclosure.

Example 1 Synthetic Scheme for Compounds 1-4

Synthesis of Compound 8

To a solution of resorufin (2.13 g, 10 mmol) in 50 mL of anhydrousdimethylforamide was added sodium hydride (437 mg, 11 mmol, 60%dispersion in mineral oil) at 0° C. After being stirred at 0° C. forhalf an hour, to the solution was then added N-phenylbis-trifluoromethane sulfonimde (4.3 g, 12 mmol). The resulting mixturewas stirred at room temperature overnight and then quenched with water.After that 1N of hydrochloric acid was added to the mixture to acidifythe solution to pH 2. Ethyl acetate was then added. The organic layerwas separated and washed with brine, dried over anhydrous sodium sulfateand evaporated in vacuo. The residue was purified by silica gel columnchromatography to give Compound 8.

Synthesis of Compound 1

An oven-dried Schlenk tube was charged with palladium (II) acetate (2mg, 1% mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (9 mg,1.5% mmol) and cesium carbonate (Cs₂CO₃) (91 mg, 0.28 mmol), and flushedwith argon gas for 5 minutes. A solution of Compound 8 (69 mg, 0.2 mmol)and 4-(methoxymethoxy)aniline (37 mg, 0.24 mmol) in toluene (2 mL) wasadded, and the resulting mixture was first stirred under argon gas atroom temperature for 30 minutes and then at 100° C. for 20 hours. Thereaction mixture was allowed to cool to room temperature, diluted withdichloromethane and filtered through a pad of Celite. The filter cakewas washed three times with 10 mL of dichloromethane. The filtrate wasthen concentrated and the residue was purified by silica gel columnchromatography to give Compound 1.

Synthesis of Compound 2

To a solution of Compound 1 (35 mg, 0.1 mmol) in dry dichloromethane (1mL) was added trifluoroacetic acid (1 mL) dropwise at 0° C. Theresulting solution was stirred at room temperature until the thin layerchromatography indicated that all starting material was consumed. Themixture was then concentrated under vacuo and azeotroped with toluenethree times. The residue was purified by silica gel columnchromatography to give Compound 2.

Synthesis of Compounds 3 and 4

Compounds 3 and 4 can be synthesized in a similar scheme as shown forCompounds 1 and 2, including a triflation reaction and a followingamination reaction.

Example 2 Synthetic Scheme for Compounds 5-7

Synthesis of Compound 9

To a solution of naphthofluorescein (4.3 g, 10 mmol) in 50 mL ofanhydrous dimethylforamide was added sodium hydride (437 mg, 11 mmol,60% dispersion in mineral oil) at 0° C. After being stirred at 0° C. forhalf an hour, the solution was then added methoxymethyl chloride (MOMCl)(0.76 mL, 10 mmol). The resulting mixture was stirred at roomtemperature overnight and then quenched with water. After that 1N ofhydrochloride was added to the mixture to acidify the solution to pH 2.Ethyl acetate was then added. The organic layer was separated and washedwith brine, dried over anhydrous sodium sulfate and evaporated in vacuo.The residue was purified by silica gel column chromatography to giveCompound 9.

Synthesis of Compound 10

To a solution of Compound 9 (476 mg, 1 mmol) and pyridine (0.32 mL, 4mmol) in dry dichloromethane under argon gas was addedtrifluoromethanesulfonic anhydride (0.34 mL, 2 mmol) dropwise at 0° C.The resulting solution was stirred at room temperature for two hours andthen quenched with water. Dichloromethane was added to the mixture andthe organic layer was separated, washed with 1N of hydrochloric acidfollowed by water and brine. The organic layer was then dried overanhydrous sodium sulfate and concentrated. The residue was purified bysilica gel column chromatography to give Compound 10.

Synthesis of Compound 11

An oven-dried Schlenk tube was charged with palladium (II) acetate (2mg, 1% mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (9 mg,1.5% mmol) and cesium carbonate (Cs₂CO₃) (91 mg, 0.28 mmol), and flushedwith argon gas for 5 minutes. A solution of Compound 10 (122 mg, 0.2mmol) and 4-(methoxymethoxy)aniline (37 mg, 0.24 mmol) in toluene (2 mL)was added, and the resulting mixture was first stirred under argon gasat room temperature for 30 minutes and then at 100° C. for 20 hours. Thereaction mixture was allowed to cool to room temperature, diluted withdichloromethane and filtered through a pad of Celite. The filter cakewas washed three times with 10 mL of dichloromethane. The filtrate wasthen concentrated and the residue was purified by silica gel columnchromatography to give Compound 11.

Synthesis of Compound 5

To a solution of Compound 11 (61 mg, 0.1 mmol) in dry dichloromethane (1mL) was added trifluoroacetic acid (1 mL) dropwise at 0° C. Theresulting solution was stirred at room temperature until the thin layerchromatography indicated that all starting material was consumed. Themixture was then concentrated under vacuo and azeotroped with toluenethree times. The residue was purified by silica gel columnchromatography to give Compound 5.

Synthesis of Compounds 6 and 7

Compounds 6 and 7 can be synthesized in a similar scheme as shown forCompound 5, including a triflation reaction and a following aminationreaction.

Example 3 Evaluation of the Fluorescence of Quenching Compounds

Each of Compounds 1-7 obtained in Example 1 and 2 was dissolved in DMFto a concentration of 10 mM, and then the solution was diluted to 10 μMby 0.1 M phosphate buffer (pH 7.4). The fluorescence spectrum of the 10μM solution of the compound was measured using a Hitachi F2500fluorescence spectrometer and the photomultiplier voltage was set to be700 V. The slit width was 2.5 nm for both excitation and emission. Themeasurement was carried out at an excitation wavelength of 600 nm. Theresults indicate that the absolute values of fluorescence intensity forCompounds 1-7 are all below 10. Therefore, Compounds 1-7 are thought tobe virtually non-fluorescent.

Example 4 Synthetic Schemes for Compound 10

Synthesis of Compound 15

To a solution of 2, 7-dichlorofluorescein (1.0 g, 2.5 mmol) andpotassium carbonate (860 mg, 6.2 mmol) in dimethylformamide (DMF) wasadded chloromethyl methyl ether (0.57 mL, 7.5 mmol). After stirring atroom temperature for 3 hours, the reaction mixture was diluted withethyl acetate and then washed with 1N of hydrochloride solution, waterand brine. The organic layer was dried over anhydrous sodium sulfate andconcentrated to give Compound 15, which is a red solid.

Synthesis of Compound 16

The red solid of Compound 15 was dissolved in a mixture oftetrahydrofuran (30 mL) and water (10 mL) containing sodium hydroxide (3g, 7.5 mmol). The solution was heated to reflux for 1 hour. Aftercooling to room temperature, the reaction solution was neutralized with1N of hydrochloride to pH 2, and then extracted with ethyl acetate. Theorganic layer was washed with brine, dried over anhydrous sodium sulfateand evaporated in vacuo. The residue was purified by silica gel columnchromatography to give Compound 16 (830 mg, 75% yield). Example 16 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 8.07 (d, J=6.7 Hz, 1H), 7.75-7.67 (m, 2H), 7.17 (d, J=7.0 Hz,1H), 7.11 (s, 1H), 6.91 (s, 1H), 6.73 (d, J=9.6 Hz, 2H), 6.44 (br, 1H),5.33-5.28 (m, 2H), 3.53 (s, 3H); ¹³C NMR (75.5 MHz, CDCl₃) δ 169.2,154.3, 153.4, 152.1, 151.0, 150.5, 135.6 (CH), 130.4 (CH), 128.7 (CH),128.0 (CH), 126.2, 125.5 (CH), 123.9 (CH), 119.1, 116.3, 112.6, 112.0,104.2 (CH), 104.1 (CH), 95.0 (CH₂), 82.5, 56.6 (CH₃); LRMS (EI) m/z (%)444 (M⁺; 5), 355 (100); and HRMS (EI) for C₂₂H₁₄Cl₂O₆: the calculatedmolecular weight was 444.0167, and the found molecular weight was444.0170.

Synthesis of Compound 17

To a solution of Compound 16 (830 mg, 1.87 mmol) and pyridine (0.6 mL,7.5 mmol) in dry dichloromethane (CH₂Cl₂) under argon gas was addedtrifluoromethanesulfonic anhydride (0.63 mL, 3.74 mmol) dropwise at 0°C. The resulting solution was stirred at room temperature for two hoursand then quenched with water. Dichloromethane was added to the mixtureand the organic layer was separated, washed with 1N of hydrochloridefollowed by water and brine. The organic layer was then dried overanhydrous sodium sulfate and concentrated. The residue was purified bysilica gel column chromatography to give Compound 17, which is a whitesolid (1.06 g, 98% yield). Compound 17 was characterized by thefollowing spectroscopic data: ¹H NMR (400 MHz, CDCl₃) δ8.09 (d, J=7.2Hz, 1H), 7.79-7.70 (m, 2H), 7.35 (s, 1H), 7.21-7.17 (m, 2H), 6.95 (s,1H), 6.80 (s, 1H), 6.44 (br, 1H), 5.34-5.29 (m, 2H), 3.53 (s, 3H); ¹³CNMR (100 MHz, CDCl₃) δ168.4, 154.6, 151.6, 150.0, 149.8, 146.1, 135.8(CH), 130.7 (CH), 130.0 (CH), 128.6 (CH), 125.7 (CH), 123.7 (CH), 122.0,120.4, 120.1, 119.9, 118.5 (q, J_(C-F)=319.0 Hz), 112.2 (CH), 112.0,104.1 (CH), 95.1 (CH₂), 80.3, 56.5 (CH₃); ¹⁹F NMR (377 MHz, CDCl₃)δ-73.1; LRMS (EI) m/z (%) 577 (M⁻; 20), 400 (100); and HRMS (EI) forC₂₃H₁₃Cl₂F₃O₈S: the calculated molecular weight was 575.9660, and thefound molecular weight was 575.9660.

Synthesis of Compound 18

An oven-dried Schlenk tube was charged with palladium (II) acetate (6mg, 2.5% mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (24mg, 3.75% mmol) and cesium carbonate (Cs₂CO₃) (228 mg, 0.7 mmol), andflushed with argon gas for 5 minutes. A solution of Compound 17 (289 mg,0.5 mmol) and 3-(4-(methylamino)phenyl)propionic acid methyl ester (116mg, 0.6 mmol) in toluene (5 mL) was added. The resulting mixture wasfirst stirred under argon gas at room temperature for 30 minutes andthen at 100° C. for 20 hours. The reaction mixture was allowed to coolto room temperature, diluted with dichloromethane and filtered through apad of Celite. The filter cake was washed three times with 10 mL ofdichloromethane. The filtrate was then concentrated and the residue waspurified by silica gel column chromatography to give Example 18 (264 mg,85% yield). Compound 18 was characterized by the following spectroscopicdata: ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J=7.5 Hz, 1H), 7.77-7.67 (m,2H), 7.23 (d, J=7.5 Hz, 1H), 7.17 (s, 1H), 7.11 (s, 1H), 7.06 (d, J=8.4Hz, 2H), 6.82 (s, 1H), 6.78 (s, 1H), 6.67 (d, J=8.4 Hz, 2H), 5.30 (m,2H), 3.66 (s, 3H), 3.52 (s, 3H), 3.26 (s, 3H), 2.88 (t, J=7.8 Hz, 2H),2.59 (t, J=7.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.4, 168.7, 154.4,151.9, 150.5, 150.4, 148.1, 146.6, 135.5, 131.7, 130.4, 129.6, 128.9,128.7, 127.1, 126.2, 125.5, 123.9, 119.1, 116.6, 116.2, 116.1, 112.5,104.1, 95.0, 81.4, 56.5, 51.5, 39.8, 35.9, 30.0; LRMS (EI) m/z (%) 619(M⁺; 9), 540 (42), 136 (100); and HRMS (EI) for C₃₃H₂₇Cl₂NO₇: thecalculated molecular weight was 619.1165, and the found molecular weightwas 619.1188.

Synthesis of Compound 19

To a solution of Compound 18 (264 mg, 0.43 mmol) in tetrahydrofuran (6mL) and water (2 mL) was added lithium hydroxide (95 mg, 2.2 mmol) at 0°C. The reaction mixture was stirred at 0° C. until all starting materialwas consumed. After that the mixture was acidified with 1 N ofhydrochloride. The solution was saturated with sodium chloride andextracted three times with 15 mL of ethyl acetate. The combined organiclayer was dried over anhydrous sodium sulfate and concentrated. Theresidue was purified by silica gel column chromatography to giveCompound 19 (237 mg, 92% yield). Example 19 was characterized by thefollowing spectroscopic data: ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J=7.5Hz, 1H), 7.77-7.68 (m, 2H), 7.23 (d, J=7.5 Hz, 1H), 7.17 (s, 1H), 7.11(s, 1H), 7.07 (d, J=8.4 Hz, 2H), 6.83 (s, 1H), 6.78 (s, 1H), 6.67 (d,J=8.4 Hz, 2H), 5.29 (m, 2H), 3.51 (s, 3H), 3.26 (s, 3H), 2.88 (t, J=7.5Hz, 2H), 2.64 (t, J=7.5 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 178.8,168.8, 154.4, 151.9, 150.5, 150.4, 148.1, 146.6, 135.5, 131.3, 130.4,129.6, 128.9, 128.7, 127.2, 126.2, 125.5, 123.9, 119.1, 116.6, 116.3,116.0, 112.5, 104.1, 95.0, 81.5, 56.5, 39.8, 35.7, 29.7; LRMS (FAB) m/z(%) 607 (M+H⁺; 8), 570 (35), 219 (100); and HRMS (EI) for C₃₂H₂₅ClNO₇(M⁺−Cl): the calculated molecular weight was 570.1320, and the foundmolecular weight was 570.1307.

Synthesis of Compound 20

To a solution of Compound 19 (237 mg, 0.39 mmol) in dry dichloromethane(3 mL) was added trifluoroacetic acid (3 mL) dropwise at 0° C. Theresulting solution was stirred at room temperature until the thin layerchromatography indicated that all starting material was consumed. Themixture was then concentrated under vacuo and azeotroped with toluenethree times to give Example 20 (250 mg, 100% yield), which was directlysubjected into the next step without any further purifications. Compound20 was characterized by the following spectroscopic data: ¹H NMR (400MHz, CD₃OD) δ 8.05 (d, J=7.6 Hz, 1H), 7.83-7.72 (m, 2H), 7.26 (d, J=7.6Hz, 1H), 7.19 (s, 1H), 7.05 (d, J=8.6 Hz, 2H), 6.84 (s, 1H), 6.77 (s,1H), 6.66 (s, 1H), 6.65 (d, J=8.6 Hz, 2H), 3.24 (s, 3H), 2.81 (t, J =7.6Hz, 2H), 2.53 (t, J=7.6 Hz, 2H).

Synthesis of Compound 21

To a solution of Compound 20 (250 mg, 0.39 mmol) in pyridine (8 mL) wasadded acetic anhydride (3 mL) and 4-dimethylaminopyridine (DMAP) (10 mg,0.08 mmol). The resulting mixture was heated to reflux for 2 hours. Thenthe reaction mixture was quenched with water and diluted with ethylacetate. The organic solution was washed with saturated sodiumbicarbonate (NaHCO₃) and brine, dried over anhydrous sodium sulfate andconcentrated. The residue was purified by silica gel columnchromatography to give Compound 21 (172 mg, 73% yield). Compound 21 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 8.07 (d, J=7.5 Hz, 1H), 7.74-7.68 (m, 2H), 7.26 (d, J=7.5 Hz,1H), 7.19 (s, 1H), 7.12 (s, 1H), 7.08 (d, J=8.3 Hz, 2H), 6.87 (s, 1H),6.84 (s, 1H), 6.68 (d, J=8.3 Hz, 2H), 3.26 (s, 3H), 2.89 (t, J=7.8 Hz,2H), 2.64 (t, J=7.8 Hz, 2H), 2.37 (s, 3H); ¹³C NMR (75.5 MHz, CDCl₃) δ178.0, 168.6, 168.0, 151.7, 150.2, 150.0, 148.4, 148.3, 146.6, 135.7(CH), 131.5, 130.6 (CH), 129.6 (CH), 129.0 (CH), 128.9 (CH), 127.5,126.0, 125.7 (CH), 124.0 (CH), 122.4, 117.8, 116.4, 116.3 (CH), 116.2(CH), 112.7 (CH), 80.9, 39.9 (CH₃), 35.6 (CH₂), 29.8 (CH₂), 20.6 (CH₃);LRMS (EI) m/z (%) 569/568 (M⁺−Cl; 10), 482 (40), 219 (100); and HRMS(EI) for C₃₂H₂₃ClNO₇ (M⁺−Cl): the calculated molecular weight was568.1163, and the found molecular weight was 568.1160.

Synthesis of Compound 22

To a solution of Compound 21 (172 mg, 0.28 mmol) in dry dichloromethane(4 mL) was added oxalyl chloride (0.12 mL, 1.4 mmol), and stirred atroom temperature for 2 hours. Then the solvent and excess oxalylchloride were evaporated off under reduced pressure. The resulting acidchloride was re-dissolved in dry dichloromethane (10 mL). To the abovesolution at −40° C. under argon gas was added trifluoroacetic anhydride(0.24 mL, 1.7 mmol) and pyridine (0.17 mL, 2.2 mmol) successively. Theresulting mixture was allowed to warm slowly to −20° C. and stirred atthat temperature for 4 hours. After that the reaction was quenched byslow addition of water (5 mL). The mixture was then diluted withdichloromethane, washed with brine, dried over anhydrous sodium sulfateand concentrated. The resulting residue was purified by silica gelcolumn chromatography to give Compound 22 (116 mg, 63% yield). Compound22 was characterized by the following spectroscopic data: ¹H NMR (400MHz, CDCl₃) δ 8.09 (d, J=7.5 Hz, 1H), 7.79-7.69 (m, 2H), 7.26 (d, J=7.5Hz, 1H), 7.19 (s, 1H), 7.12 (s, 1H), 7.05 (d, J=8.6 Hz, 2H), 6.87 (s,1H), 6.85 (s, 1H), 6.67 (d, J=8.6 Hz, 2H), 3.27 (s, 3H), 3.01 (t, J=7.3Hz, 0.85×2H, —COCF₃), 2.92 (t, J=7.3 Hz, 0.85×2H, —COCF₃), 2.82 (t,J=7.3 Hz, 0.15×2H, —C(OH)₂CF₃), 2.37 (s, 3H), 2.13 (t, J=7.3 Hz,0.15×2H, —C(OH)₂CF₃); ¹³C NMR (75.5 MHz, CDCl₃) δ 190.5 (q, J=35.2 Hz),168.5, 167.9, 151.7, 150.3, 150.0, 148.4, 148.2, 146.9, 135.7, 130.6,130.4, 129.7, 129.0, 128.9, 127.6, 126.0, 125.7, 124.0, 122.4, 117.8,116.6, 116.5, 116.1, 115.8 (q, J_(C-F)=297.7), 112.7, 80.8, 39.8, 38.3,27.5, 20.6; LRMS (EI) m/z (%) 656 (M⁺; 17), 534 (100); and HRMS (EI) forC₃₃H₂₂Cl₂F₃NO₆: the calculated molecular weight was 655.0776, and thefound molecular weight was 655.0783.

Synthesis of Compound 10

To a solution of Compound 22 (66 mg, 0.1 mmol) in methanol (3 mL) wasadded a solution of potassium carbonate (41 mg, 0.3 mmol) in water (1mL). After stirring at room temperature for 3 hours, the resultingmixture was diluted with ethyl acetate, washed with diluted hydrochloricacid and brine. The organic solution was then dried over anhydroussodium sulfate and concentrated. The residue was purified by silica gelcolumn chromatography to give Compound 10 (60 mg, 98% yield). Compound10 was characterized by the following spectroscopic data: ¹H NMR (400MHz, CDCl₃) δ 8.08 (d, J=7.5 Hz, 1H), 7.79-7.69 (m, 2H), 7.25 (d, J=7.5Hz, 1H), 7.18 (s, 1H), 7.05 (d, J=8.3 Hz, 2H), 6.92 (s, 1H), 6.82 (s,1H), 6.74 (s, 1H), 6.68 (d, J=8.3 Hz, 2H), 3.26 (s, 3H), 3.01 (t, J=7.0Hz, 2H), 2.91 (t, J=7.5 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 190.9 (q,J=35.0 Hz), 169.0, 153.5, 151.9, 151.0, 150.5, 148.0, 146.9, 135.7,130.5, 130.3, 129.7, 128.9, 128.0, 127.2, 126.2, 125.6, 123.9, 116.7,116.5, 116.1, 116.3, 115.5 (q, J_(C-F)=290.3), 112.0, 104.2, 81.7, 39.8,38.3, 27.4;LRMS (EI) m/z (%) 614 (M⁺; 16), 535 (100); and HRMS (EI) forC₃₁H₂₀Cl₂F₃NO₅: the calculated molecular weight was 613.0671, and thefound molecular weight was 613.0682.

Example 5 Synthetic Schemes for Compounds 12 and 12a

Synthesis of Compound 23

To a solution of fluorescein (3.3 g, 10 mmol) in 50 mL of anhydrousdimethylforamide was added sodium hydride (437 mg, 11 mmol, 60%dispersion in mineral oil) at 0° C. After being stirred at 0° C. forhalf an hour, the solution was then added methoxymethyl chloride (MOMCl)(0.76 mL, 10 mmol). The resulting mixture was stirred at roomtemperature overnight and then quenched with water. After that 1N ofhydrochloride was added to the mixture to acidify the solution to pH 2.Ethyl acetate was then added. The organic layer was separated and washedwith brine, dried over anhydrous sodium sulfate and evaporated in vacuo.The residue was purified by silica gel column chromatography to giveCompound 23 (3.2 g, 85% yield). Compound 23 was characterized by thefollowing spectroscopic data: ¹H NMR (400 MHz, CDCl₃) δ 8.01 (d, J=7.6Hz, 1H), 7.66-7.59 (m, 2H), 7.14 (d, J=7.6 Hz, 1H), 6.92 (d, J=1.7 Hz,1H), 6.73 (d, J=1.9 Hz, 1H), 6.68-6.67 (m, 2H), 6.55-6.54 (m, 2H), 5.18(s, 2H), 3.46 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.6, 158.7, 158.6,152.8, 152.4, 152.3, 135.3 (CH), 129.8 (CH), 129.0 (CH), 128.9 (CH),126.5, 125.0 (CH), 124.0 (CH), 112.9 (CH), 112.6 (CH), 112.0, 110.2,103.5 (CH), 103.1 (CH), 94.1 (CH₂), 85.2, 56.1 (CH₃); LRMS (EI) m/z (%)376 (M⁺; 7), 332 (100); and HRMS (EI) for C₂₂H₁₆O₆: the calculatedmolecular weight was 376.0947, and the found molecular weight was376.0949.

Synthesis of Compound 24

To a solution of Compound 23 (3.2 g, 8.5 mmol) and pyridine (2.74 mL, 34mmol) in dry dichloromethane under argon gas was addedtrifluoromethanesulfonic anhydride (2.86 mL, 17 mmol) dropwise at 0° C.The resulting solution was stirred at room temperature for two hours andthen quenched with water. Dichloromethane was added to the mixture andthe organic layer was separated, washed with 1N of hydrochloridefollowed by water and brine. The organic layer was then dried overanhydrous sodium sulfate and concentrated. The residue was purified bysilica gel column chromatography to give Compound 24 (4.2 g, 98% yield).Compound 24 was characterized by the following spectroscopic data: ¹HNMR (400 MHz, CDCl₃) δ 8.04 (d, J=7.5 Hz, 1H), 7.72-7.65 (m, 2H), 7.27(d, J=2.3 Hz, 1H), 7.18 (d, J=7.4 Hz, 1H), 7.01-6.93 (m, 3H), 6.77-6.73(m, 2H), 5.19 (s, 2H), 3.46 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 168.7,159.0, 152.3, 151.9, 151.5, 149.8, 135.3 (CH), 130.1 (CH), 129.9 (CH),128.8 (CH), 126.0, 125.1 (CH), 123.7 (CH), 123.0, 120.1, 119.7, 116.9,116.5 (CH), 113.6 (CH), 111.6, 110.3 (CH), 103.5 (CH), 94.1 (CH₂), 81.3,55.9 (CH₃); ¹⁹F NMR (377 MHz, CDCl₃) δ-72.7; LRMS (EI) m/z (%) 508 (M⁻;23), 331 (100); and HRMS (EI) for C₂₃H₁₅F₃O₈S: the calculated molecularweight was 508.0440, and the found molecular weight was 508.0438.

Synthesis of Compound 25

An oven-dried Schlenk tube was charged with palladium (II) acetate (2mg, 1% mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (9 mg,1.5% mmol) and cesium carbonate (Cs₂CO₃) (91 mg, 0.28 mmol), and flushedwith argon gas for 5 minutes. A solution of Compound 24 (102 mg, 0.2mmol) and 4-(methoxymethoxy)aniline (37 mg, 0.24 mmol) in toluene (2 mL)was added, and the resulting mixture was first stirred under argon gasat room temperature for 30 minutes and then at 100° C. for 20 hours. Thereaction mixture was allowed to cool to room temperature, diluted withdichloromethane and filtered through a pad of Celite. The filter cakewas washed three times with 10 mL of dichloromethane. The filtrate wasthen concentrated and the residue was purified by silica gel columnchromatography to give Compound 25 (84 mg, 82% yield). Compound 25 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 7.99 (d, J=7.4 Hz, 1H), 7.66-7.58 (m, 2H), 7.16 (d, J=7.4 Hz,1H), 7.08 (d, J=8.9 Hz, 2H), 6.99 (d, J=8.9 Hz, 2H), 6.91 (s, 1H), 6.73(s, 1H), 6.67 (s, 2H), 6.57-6.48 (m, 2H), 5.94 (s, br, 1H), 5.16 (s,2H), 5.14 (s, 2H), 3.48 (s, 3H), 3.45 (s, 3H); ¹³C NMR (75.5 MHz, CDCl₃)δ 169.6, 158.8, 153.5, 153.1, 152.6, 152.5, 147.6, 135.3, 134.9, 129.6,129.1, 129.0, 127.0, 124.9, 124.0, 123.2, 117.4, 112.7, 112.6, 111.9,109.0, 103.6, 100.8, 94.9, 94.3, 83.8, 56.1, 56.0; LRMS (EI) m/z (%) 511(M⁺; 47), 467 (100); and HRMS (EI) for C₃₀H₂₅NO₇: the calculatedmolecular weight was 511.1631, and the found molecular weight was511.1632.

Synthesis of Compound 25

To a solution of Compound 25 (84 mg, 0.16 mmol) in tetrahydrofuran (4mL) at 0° C. was added sodium hydride (10 mg, 0.24 mmol, 60% in mineraloil). The suspension was stirred for half an hour and then methyl iodide(20 μL, 0.32 mmol) was introduced. The mixture was stirred at roomtemperature overnight and then quenched with water. The mixture wasdiluted with ethyl acetate, washed with 1N hydrochloric acid and brine.After dried over anhydrous sodium sulfate the organic solution wasconcentrated in vacuo and the residue was purified by silica gel columnchromatography to give Compound 26 (64 mg, 76% yield). Compound 26 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 7.98 (d, J=7.2 Hz, 1H), 7.66-7.58 (m, 2H), 7.15 (d, J=7.2 Hz,1H), 7.14-7.08 (m, 2H), 7.04 (d, J=9.0 Hz, 2H), 6.92 (s, 1H), 6.67 (s,1H), 6.52 (d, J=9.0 Hz, 2H), 6.36-6.34 (m, 2H), 5.16 (s, 4H), 3.48 (s,3H), 3.45 (s, 3H), 3.25 (s, 3H); ¹³C NMR (75.5 MHz, CDCl₃) δ 169.5,158.7, 155.0, 153.1, 152.6, 152.4, 151.5, 141.7, 134.8, 129.5, 129.1,128.4, 127.7, 127.1, 124.8, 124.0, 117.5, 112.8, 112.6, 110.9, 107.5,103.6, 100.5, 94.6, 94.3, 83.8, 56.1, 56.0, 40.4; LRMS (EI) m/z (%) 526(M⁺; 8), 482 (100); and HRMS (EI) for C₃₁H₂₇NO₇: the calculatedmolecular weight was 525.1788, and the found molecular weight was525.1792.

Synthesis of Compound 12

To a solution of Compound 26 (64 mg, 0.12 mmol) in dry dichloromethane(CH₂Cl₂) (2 mL) was added trifluoroacetic acid (2 mL) dropwise at 0° C.The resulting solution was stirred at room temperature until the thinlayer chromatography indicated that all starting material was consumed.The mixture was then concentrated in vacuo and azeotroped with toluenethree times. The residue was dissolved in ethyl acetate and washed withsaturated sodium bicarbonate (NaHCO₃), followed by water and brine. Theorganic solution was concentrated in vacuo and then the residue waspurified by silica gel column chromatography to give Compound 12 (47 mg,90% yield). Compound 12 was characterized by the following spectroscopicdata: ¹H NMR (500 MHz, CD₃OD) δ 8.26 (d, J=7.7 Hz, 1H), 7.83-7.75 (m,2H), 7.35 (d, J=7.7 Hz, 1H), 7.13 (d, J=8.7 Hz, 2H), 7.09 (d, J=9.0 Hz,1H), 7.04-7.02 (m, 2H), 6.92-6.88 (m, 4H), 6.80 (d, J =9.4 Hz, 1H), 3.52(s, 3H); ¹³C NMR (125.8 MHz, CD₃OD) δ 169.1, 168.2, 159.3, 159.1, 158.7,158.0, 139.2, 138.1, 134.6, 132.3, 132.1, 131.7, 131.6, 131.3, 130.1,130.0, 128.9, 118.1, 118.0, 117.4, 115.9, 115.4, 103.6, 99.1, 42.3; LRMS(EI) m/z (%) 437 (M⁺; 6), 393 (100); and HRMS (EI) for C₂₇H₁₉NO₅: thecalculated molecular weight was 437.1263, and the found molecular weightwas 437.1266.

Synthesis of Compound 12a

To a solution of Compound 12 (108 mg, 0.25 mmol) in dry dichloromethane(CH₂Cl₂) (4 mL) was added pyridine (0.4 mL) and acetyl chloride (0.8 mL)successively. The resulting solution was stirred at room temperatureuntil thin layer chromatography indicated that all starting material wasconsumed. The reaction was then quenched by saturated NH₄Cl solution andextracted with ethyl acetate. The organic solution was concentrated invacuo and then the residue was purified by silica gel columnchromatography to give Compound 12a (110 mg, 85% yield). Compound 12awas characterized by the following spectroscopic data: ¹H NMR (400 MHz,CDCl₃) δ 8.00 (d, J=7.2 Hz, 1H), 7.66-7.60 (m, 2H), 7.19-7.17 (m, 3H),7.08 (d, J=9.0 Hz, 2H), 7.02 (d, J=1.8 Hz, 1H), 7.78-7.76 (m, 2H), 6.66(d, J=1.8 Hz, 1H), 6.56 (d, J=9.0 Hz, 1H), 6.48 (dd, J=9.0, 1.8 Hz, 1H),3.31 (s, 3H), 2.30 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 169.5, 169.3,168.9, 152.9, 152.2, 152.1, 151.8, 151.0, 147.5, 145.2, 135.0, 129.7,129.0, 128.5, 126.8, 126.3, 125.0, 124.1, 122.8, 117.2, 116.9, 112.4,110.2, 108.4, 102.1, 83.0, 40.4, 21.1; LRMS (EI) m/z (%) 521 (M⁺; 26),477 (100); and HRMS (EI) for C₃₁H₂₃NO₇: the calculated molecular weightwas 521.1475, and the found molecular weight was 521.1471

Example 6 Synthetic Schemes for Compound 14

Synthesis of Compound 27

An oven-dried Schlenk tube was charged with palladium (II) acetate (2mg, 1% mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (9 mg,1.5% mmol) and cesium carbonate (Cs₂CO₃) (91 mg, 0.28 mmol), and flushedwith argon gas for 5 minutes. A solution of Compound 24 (102 mg, 0.2mmol) and 4-nitroaniline (33 mg, 0.24 mmol) in toluene (2 mL) was added,and the resulting mixture was first stirred under argon gas at roomtemperature for 30 minutes and then at 100° C. for 20 hours. Thereaction mixture was allowed to cool to room temperature, diluted withdichloromethane and filtered through a pad of Celite. The filter cakewas washed three times with 10 mL of dichloromethane. The filtrate wasthen concentrated and the residue was purified by silica gel columnchromatography to give Compound 27 (79 mg, 80% yield). Compound 27 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 8.06-8.01 (m, 3H), 7.72-7.63 (m, 2H), 7.19 (d, J=7.2 Hz, 1H),7.06-7.03 (m, 3H), 6.93 (s, 1H), 6.80-6.64 (m, 4H), 5.18 (s, 2H), 3.46(s, 3H); ¹³C NMR (75.5 MHz, CDCl₃) δ 169.8, 159.0, 152.8, 152.2, 148.8,142.6, 140.3, 135.4, 130.0, 129.1, 129.0, 126.5, 126.0, 125.1, 124.0,115.9, 115.1, 113.2, 112.0, 1069, 1037, 943, 834, 56.2; LRMS (FAB) m/z(%) 496 (M⁺; 20), 154(100); and HRMS (EI) for C₂₇H₂₀N₂O₅ (M⁺−CO₂): thecalculated molecular weight was 452.1372, and the found molecular weightwas 452.1366.

Synthesis of Compound 28

To a solution of Compound 27 (79 mg, 0.16 mmol) in tetrahydrofuran (4mL) at 0° C. was added sodium hydride (10 mg, 0.24 mmol, 60% in mineraloil). The suspension was stirred for half an hour and then methyl iodide(20 μL, 0.32 mmol) was introduced. The mixture was stirred at roomtemperature overnight and then quenched with water. The mixture wasdiluted with ethyl acetate, washed with 1N hydrochloric acid and brine.After dried over anhydrous sodium sulfate the organic solution wasconcentrated in vacuo and the residue was purified by silica gel columnchromatography to give Compound 28 (67 mg, 83% yield). Compound 28 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 8.10-8.04 (m, 3H), 7.80-7.63 (m, 2H), 7.24 (d, J=7.4 Hz, 1H),7.14 (d, J=2.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.90-6.82 (m, 4H),6.76-6.73 (m, 2H), 5.20 (s, 2H), 3.48 (s, 3H), 3.44 (s, 3H); ¹³C NMR(75.5 MHz, CDCl₃) δ 159.0, 153.0, 152.7, 152.4, 152.1, 148.5, 139.4,135.2, 130.0, 129.6, 129.1, 126.6, 125.7, 125.2, 124.0, 120.8, 116.5,114.5, 113.3 (2C), 112.1, 103.7, 94.4, 85.3, 56.2, 40.4; LRMS (FAB) m/z(%) 510 (M⁺; 20), 109 (100); and HRMS (EI) for C₂₈H₂₂N₂O₅ (M⁺−CO₂): thecalculated molecular weight was 466.1522, and the found molecular weightwas 466.1529.

Synthesis of Compound 29

To a solution of Compound 28 (67 mg, 0.13 mmol) in ethanol (10 mL) wasslowly added palladium (10% on activated carbon powder, 7 mg). Themixture was hydrogenated for 2 hours at room temperature. The mixturewas then filtered through a pad of Celite, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography to give Compound 29 (48 mg, 77% yield). Compound 29 wascharacterized by the following spectroscopic data: ¹H NMR (300 MHz,CDCl₃) δ 7.99 (d, J=7.5 Hz, 1H), 7.64-7.57 (m, 2H), 7.17 (d, J =7.5 Hz,1H), 6.98 (d, J=8.6 Hz, 2H), 6.91 (s, 1H), 6.710-6.67 (m, 4H), 6.51-6.48(m, 2H), 6.35-6.32 (m, 1H), 5.18 (s, 2H), 3.68 (br, 2H), 3.47 (s, 3H),3.24 (s, 3H); ¹³C NMR (75.5 MHz, CDCl₃) δ 169.6, 158.7, 153.2, 152.7,152.4, 151.8, 144.5, 138.7, 134.7, 129.4, 129.1, 128.3, 128.0, 127.2,124.8, 124.0, 116.2, 112.8, 112.5, 110.5, 106.8, 103.6, 99.8, 94.4,84.0, 56.1, 40.4; LRMS (EI) m/z (%) 481 (M⁺; 24), 437 (100); and HRMS(EI) for C₂₉H₂₄N₂O₅: the calculated molecular weight was 480.1685, andthe found molecular weight was 480.1688.

Synthesis of Compound 14

To a solution of Compound 29 (48 mg, 0.10 mmol) in dry dichloromethane(2 mL) was added trifluoroacetic acid (2 mL) dropwise at 0° C. Theresulting solution was stirred at room temperature until the thin layerchromatography indicated that all starting material was consumed. Themixture was then concentrated in vacuo and azeotroped with toluene threetimes. The residue was dissolved in ethyl acetate and washed withsaturated sodium bicarbonate (NaHCO₃), followed by water and brine. Theorganic solution was concentrated in vacuo and then the residue waspurified by silica gel column chromatography to give Compound 14 (40 mg,91% yield). Compound 14 was characterized by the following spectroscopicdata: ¹H NMR (400 MHz, CD₃OD) δ 8.02 (d, J=7.2 Hz, 1H), 7.70-7.64 (m,2H), 7.16 (d, J=7.2 Hz, 1H), 6.92 (d, J=8.6 Hz, 2H), 6.76 (d, J=8.6 Hz,2H), 6.69 (d, J=8.8 Hz, 1H), 6.64 (d, J=2.2 Hz, 1H), 6.59-6.52 (m, 3H),6.42 (dd, J=8.8, 2.2 Hz, 1H), 3.26 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ164.6, 154.5, 153.7, 153.6, 153.5, 147.5, 146.2, 137.3, 133.7, 129.5,129.4, 128.5, 127.6, 127.3, 125.9, 125.4, 116.1, 114.6, 114.1, 111.4,108.5, 102.3, 98.6, 76.0, 39.7; LRMS (ESI) m/z (%) 437 (M+H⁺; 100); andHRMS (EI) for C₂₇H₂₀N₂O₄: the calculated molecular weight was 436.1423,and the found molecular weight was 436.1424.

Example 7 Synthetic Schemes for Compound 30

Synthesis of Compound 31

2′-Carboxy-5-chloro-2,4-dihydroxybenzophenone and1,6-dihydroxynaphthalene were combined in methanesulfonic acid andsealed in a thich-walled glass tube. After the resulting mixture wasstirred at 90° C. for 24 hours, the reaction was poured into ice-coldwater, and the precipitate was filtered, washed with water and dried invacuo. The crude product 31 was used in the next step without furtherpurification.

Synthesis of Compound 32

To a solution of Compound 31 (1.23 g, 2.95 mmol) and potassium carbonate(407 mg, 2.95 mmol) in dimethylformamide (DMF) was added chloromethylmethyl ether (0.22 mL, 2.95 mmol). After stirring at room temperaturefor 3 hours, the reaction mixture was diluted with ethyl acetate andthen washed with 1N of hydrochloride solution, water and brine. Theorganic layer was dried over anhydrous sodium sulfate and concentrated.The residue was purified by silica gel column chromatography to giveCompound 32 (680 mg, 50% yield).

Synthesis of Compound 33

To a solution of Compound 32 (340 mg, 0.75 mmol) and pyridine (0.36 mL,4.48 mmol) in dry dichloromethane (CH₂Cl₂) under argon gas was addedtrifluoromethanesulfonic anhydride (0.38 mL, 2.24 mmol) dropwise at 0°C. The resulting solution was stirred at room temperature for two hoursand then quenched with water. Dichloromethane was added to the mixtureand the organic layer was separated, washed with 1N of hydrochloridefollowed by water and brine. The organic layer was then dried overanhydrous sodium sulfate and concentrated. The residue was purified bysilica gel column chromatography to give Compound 33 as a white solid(436 mg, 98% yield).

Synthesis of Compound 34

An oven-dried Schlenk tube was charged with palladium (II) acetate (5mg, 0.02 mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (19mg, 0.03 mmol) and cesium carbonate (Cs₂CO₃) (79 mg, 0.24 mmol), andflushed with argon gas for 5 minutes. A solution of Compound 33 (120 mg,0.2 mmol) and 4-(methoxymethoxy)-N-methylaniline (36 mg, 0.21 mmol) intoluene (3 mL) was added. The resulting mixture was first stirred underargon gas at room temperature for 30 minutes and then at 100° C. for 20hours. The reaction mixture was allowed to cool to room temperature,diluted with dichloromethane and filtered through a pad of Celite. Thefilter cake was washed three times with 10 mL of dichloromethane. Thefiltrate was then concentrated and the residue was purified by silicagel column chromatography to give Compound 34 (104 mg, 85% yield).

Synthesis of Compound 30

To a solution of Compound 34 (104 mg, 0.17 mmol) in dry dichloromethane(2 mL) was added trifluoroacetic acid (2 mL) dropwise at 0° C. Theresulting solution was stirred at room temperature until thin layerchromatography indicated that all starting material was consumed. Themixture was then concentrated in vacuo and azeotroped with toluene threetimes. The residue was dissolved in ethyl acetate and washed withsaturated sodium bicarbonate (NaHCO₃), followed by water and brine. Theorganic solution was concentrated in vacuo and then the residue waspurified by silica gel column chromatography to give Compound 30 (72 mg,82% yield). Compound 30 was characterized by the following spectroscopicdata: ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J=9.3 Hz, 1H), 8.09 (dd, J=6.8,1.1 Hz, 1H), 7.71-7.63 (m, 2H), 7.23 (d, J=8.8 Hz, 1H), 7.16 (dd, J=6.8,1.1 Hz, 1H), 7.08-7.04 (m, 3H), 6.99 (s, 1H), 6.91-6.86 (m, 3H), 6.80(s, 1H), 6.61 (dd, J=8.8 Hz, 1H), 3.33 (s, 3H), 3.18 (br, 2H); ¹³C NMR(100 MHz, CDCl₃) δ 169.7, 154.5, 152.0, 149.5, 147.5, 140.3, 136.5,135.0, 129.9, 128.4, 127.2, 126.9, 125.7, 124.6, 123.8, 122.6, 122.5,118.1, 116.4, 116.2, 111.9, 108.2, 107.3, 104.1, 40.6; LRMS (EI) m/z (%)521 (M⁺; 16), 476 (100); and HRMS (EI) for C₃₁H₂₀ClNO₅: the calculatedmolecular weight was 521.1030, and the found molecular weight was521.1033.

Example 8 Specific Detection of Peroxynitrite with Compound 10

UV-Visible Absorption Spectrum of Compound 10

Compound 10 obtained in Example 4 was dissolved in pH 7.4 0.1 Mphosphate buffer containing 0.1% DMF as cosolvent to form a 10 μMsolution. The absorption spectrum of the 10 μM solution of Compound 10was measured and showed that Compound 10 has an absorption maximum atabout 520 nm.

Emission Spectra of Compound 10

Compound 10 obtained in Example 4 was dissolved in DMF to aconcentration of 10 mM, and then the solution was diluted to 10 μM by0.1 M phosphate buffer (pH 7.4). The fluorescence spectrum of the 10 μMsolution of Compound 10 was measured using a Hitachi F2500 fluorescencespectrometer and the photomultiplier voltage was 700 V. The slit widthwas 2.5 nm for both excitation and emission. The measurement was carriedout at an excitation wavelength of 520 nm. The results shown in FIG. 1indicate that Compound 10 itself is non-fluorescent.

Detection of Peroxynitrite with Compound 10

Compound 10 obtained in Example 4 was dissolved in DMF to aconcentration of 10 mM, and then the solution was diluted to 10μM by 0.1M potassium phosphate buffer (pH 7.4). Peroxynitrite solution in 0.1 MNaOH was prepared by the method of Keith and Powell (Keith, W. G. &Powell, R. E.; Kinetics of decomposition of peroxynitrous acid; J. Chem.Soc. A, 1969, 1, 90), and its concentration in the stock solutions usedwas estimated by using an extinction coefficient of 1670 cm⁻¹ (mol/L)⁻¹at 302 nm (Hughes and Nicklin; The chemistry of pernitrites. Part I.Kinetics of decomposition of pernitrious acid; J. Chem. Soc. A, 1968, 2,450-452). Peroxynitrite stock solution was added into the solution ofCompound 10 to provide various final concentrations, like 0, 2, 6, 10,20, 30, 50 100, and 200 μM. Fluorescence spectra of the solutions weremeasured after 5 minutes under the same conditions as mentioned above.The fluorescence spectra are shown in FIG. 1. As clearly shown in FIG.1, the fluorescence intensity of Compound 10 increase significantlyafter the addition of peroxynitrite. Further, FIG. 2 shows that thefluorescence intensity at 541 nm increases linearly with increasingconcentration of peroxynitrite.

Comparison of Specificity of Compound 10 with Different ROS and RNS

The reactivity of Compound 10 was compared toward different reactiveoxygen species (ROS) and reactive nitrogen species (RNS), includingOCl⁻, H₂O₂, ¹O₂, NO, O₂ ^(•−), ^(•)OH, ONOO⁻ and alkylperoxyl radical(ROO^(•)). Different reactive oxygen species and reactive nitrogenspecies were added independently to 5 mL of the solution of Compound 10(10 μM in 0.1 M potassium phosphate buffer). The changes of fluorescenceintensity before and after the treatment were measured. The results areshown in FIG. 3. The reactive oxygen species and reactive nitrogenspecies were prepared as follows.

-   -   a. H₂O₂ (final 100 μM) was added and then stirred for 1 hour at        25° C.    -   b. (3-(1,4-Dihydro-1,4-epidioxy-1-naphthyl)propionic acid)        (final 100 μM) was added and then stirred at 25° C. for 1 hour.    -   c. 2,2′-Azobis(2-amidinopropane)dihydrochloride (final 100 μM)        was added and then stirred at 25° C. for 1 hour.    -   d. SNP (Sodium Nitroferricyanide (III) Dihydrate) (final 100 μM)        was added and then stirred for 1 hour at 25° C.    -   e. O₂ ^(•−) was generated by xanthine and xanthine oxidase.        Xanthine oxidase was added firstly. After xanthine oxidase was        dissolved, xanthine (final 100 μM) was added and the mixtures        were stirred at 25° C. for 1 hour.    -   f. Ferrous chloride (final 10 μM) was added in the presence of        10 equivalences of H₂O₂ (100 μM).    -   g. ONOO⁻ (final 10 μM) was added at 25° C.    -   h. NaOCl (final 10 μM) was added at 25° C. Commercial bleach was        the source of NaOCl.

FIG. 3 shows that peroxynitrite leads to much stronger fluorescenceenhancement of Compound 10 than any other ROS and RNS. These resultsdemonstrated that Compound 10 has a much higher reactivity towardsperoxynitrite among ROS and RNS in an abiotic system. Further, similarreactions do not proceed between the trifluoromethyl derivative ofCompound 10 and any other reactive oxygen species or reactive nitrogenspecies present in the biological systems.

Example 9 Application of Compound 22 in Cell Assay

Murine J744.1 macrophages (ATCC, USA) were used to investigate thepotential of Compound 22 (acetate form of Compound 10) for the detectionof peroxynitrite in living cells. J744.1 macrophages were cultured inDulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 10%heat-inactivated fetal bovine serum (Gibco) supplemented with 100 U/mlof penicillin and 100 μg/ml streptomycin at 37° C., 5% CO₂. They weresubcultured by scraping and seeded on six-well plates according tomanufacture's instruction. The growth medium was changed every two tothree days. Cells were grown to confluence prior to experiment. MurineJ744.1 macrophages were incubated with Compound 22 (20 μM) for 1 hr andthen washed three times with PBS buffer. Only very weak fluorescence wasobserved in the absence of stimulants (FIG. 8A). The fluorescence wasinduced after treatment with LPS (lipopolysaccharide, 1 μg/ml) and IFN-γ(Interferon-γ, 50 ng/ml) for 4 hrs (FIG. 8B) and strong fluorescence wasobserved after additional stimulation by PMA (phorbol 12-myristate13-acetate, 10 nM) for half an hour (FIG. 8C). Thus, we conclude thatCompound 22 is suitable for the detection of peroxynitrite produced instimulated Murine J744.1 macrophages.

Example 10 Highly Sensitive Detection of Peroxynitrite with Compound 12

UV-Visible Absorption Spectrum of Compound 12

Compound 12 obtained in Example 5 was dissolved in pH 7.4 0.1 Mphosphate buffer containing 0.1% DMF as cosolvent to form a 1 μMsolution. The absorption spectrum of the 1 μM solution of Compound 12was measured and showed that Compound 12 has an absorption maximum atabout 515 nm.

Emission Spectra of Compound 12

Compound 12 obtained in Example 5 was dissolved in DMF to aconcentration of 1 mM, and then the solution was diluted to 1 μM by 0.1M phosphate buffer (pH 7.4). The fluorescence spectrum of the 1 μMsolution of Compound 12 was measured using a Hitachi F2500 fluorescencespectrometer and the photomultiplier voltage was 700 V. The slit widthwas 2.5 nm for both excitation and emission. The measurement was carriedout at an excitation wavelength of 515 nm. The results shown in FIG. 4indicate that Compound 12 itself is non-fluorescent.

Detection of Peroxynitrite with Compound 12

Compound 12 obtained in Example 5 was dissolved in DMF to aconcentration of 1 mM, and then the solution was diluted to 1 μM by 0.1M potassium phosphate buffer (pH 7.4). Peroxynitrite solution in 0.1 MNaOH was prepared by the method of Keith and Powell (Keith, W. G. &Powell, R. E.; Kinetics of decomposition of peroxynitrous acid; J. Chem.Soc. A, 1969, 1, 90), and its concentration in the stock solutions usedwas estimated by using an extinction coefficient of 1670 cm⁻¹ (mol/L)⁻¹at 302 nm (Hughes and Nicklin; The chemistry of pernitrites. Part I.Kinetics of decomposition of pernitrious acid; J. Chem. Soc. A, 1968, 2,450-452). Peroxynitrite stock solution was added into the solution ofCompound 12 to provide various final concentrations, like 0, 1, 2, 3, 4,5, 6, and 7 μM. Fluorescence spectra of the solutions were measuredafter 5 minutes under the same conditions as mentioned above. Thefluorescence spectra are shown in FIG. 4. As clearly shown in FIG. 4,the fluorescence intensity of Compound 12 increase significantly afterthe addition of peroxynitrite. Further, the fluorescence intensity at535 nm increases linearly with increasing concentration of peroxynitrite(data not shown).

Comparison of Specificity of Compound 12 with Different ROS and RNS

The reactivity of Compound 12 was compared toward different reactiveoxygen species (ROS) and reactive nitrogen species (RNS), includingOCl⁻, H₂O₂, ¹O₂, NO, O₂ ^(•−), ^(•)OH, ONOO⁻ and alkylperoxyl radical(ROO^(•)). Different reactive oxygen species and reactive nitrogenspecies were added independently to 5 mL of the solution of Compound 12(1 μM in 0.1 M potassium phosphate buffer). The changes of fluorescenceintensity before and after the treatment were measured. The results areshown in FIG. 5. The reactive oxygen species and reactive nitrogenspecies were prepared as follows:

-   -   i. H₂O₂ (final 100 μM) was added and then stirred for 1 hour at        25° C.    -   j. (3-(1,4-Dihydro-1,4-epidioxy-1-naphthyl)propionic acid)        (final 100 μM) was added and then stirred at 25° C. for 1 hour.    -   k. 2,2′-Azobis(2-amidinopropane)dihydrochloride (final 100 μM)        was added and then stirred at 25° C. for 1 hour.    -   l. SNP (Sodium Nitroferricyanide (III) Dihydrate) (final 100 μM)        was added and then stirred for 1 hour at 25° C.    -   m. O₂ ^(•−) was generated by xanthine and xanthine oxidase.        Xanthine oxidase was added firstly. After xanthine oxidase was        dissolved, xanthine (final 100 μM) was added and the mixtures        were stirred at 25° C. for 1 hour.    -   n. Ferrous chloride (final 10 μM) was added in the presence of        10 equivalences of H₂O₂ (100 μM).    -   o. ONOO⁻ (final 10 μM) was added at 25° C.    -   p. NaOCl (final 10 μM) was added at 25° C. Commercial bleach was        the source of NaOCl.

FIG. 5 shows that peroxynitrite leads to much stronger fluorescenceenhancement of Compound 12 than any other ROS and RNS. These resultsdemonstrated that Compound 12 has a much higher reactivity towardsperoxynitrite among ROS and RNS in an abiotic system. Further, similarreactions do not proceed between the phenol derivative of Compound 12and any other reactive oxygen species or reactive nitrogen speciespresent in the biological systems.

Example 11 Highly Sensitive Detection of Hypochlorite with Compound 14

UV-Visible Absorption Spectrum of Compound 14

Compound 14 obtained in Example 6 was dissolved in pH 7.4 0.1 Mphosphate buffer containing 0.1% DMF as cosolvent to form a 1 μMsolution. The absorption spectrum of the 1 μM solution of Compound 14was measured and showed that Compound 14 has an absorption maximum atabout 515 nm.

Emission Spectra of Compound 14

Compound 14 obtained in Example 6 was dissolved in DMF to aconcentration of 1 mM, and then the solution was diluted to 1 μM by 0.1M phosphate buffer (pH 7.4). The fluorescence spectrum of the 1 μMsolution of Compound 14 was measured using a Hitachi F2500 fluorescencespectrometer and the photomultiplier voltage was 700 V. The slit widthwas 2.5 nm for both excitation and emission. The measurement was carriedout at an excitation wavelength of 515 nm. The results shown in FIG. 6indicate that Compound 14 itself is non-fluorescent.

Detection of Peroxynitrite with Compound 14

Compound 14 obtained in Example 6 was dissolved in DMF to aconcentration of 1 mM, and then the solution was diluted to 1 μM by 0.1M potassium phosphate buffer (pH 7.4). Commercial bleach was the sourceof NaOCl. The concentration of NaOCl was determined by titration withsodium thiosulfate solution which was standardized by the titration withKlO₃. Then NaOCl was added to provide final concentrations of 0, 2, 3,4, 5, 6, 7, and 8 μM. Fluorescence spectra of the solutions weremeasured after 5 minutes under the same conditions as mentioned above.The fluorescence spectra are shown in FIG. 6. As clearly shown in FIG.6, the fluorescence intensity of Compound 14 increase significantlyafter the addition of hypochlorite. Further, the fluorescence intensityat 535 nm increases linearly with increasing concentration ofhypochlorite.

Comparison of Specificity of Compound 14 with Different ROS and RNS

The reactivity of Compound 14 was compared toward different reactiveoxygen species (ROS) and reactive nitrogen species (RNS), includingOCl⁻, H₂O₂, ¹O₂, NO, O₂ ^(•−), ^(•)OH, ONOO⁻ and alkylperoxyl radical(ROO^(•)). Different reactive oxygen species and reactive nitrogenspecies were added independently to 5 mL of the corresponding solutionof Compound 14 (1 μM in 0.1 M potassium phosphate buffer). The changesin fluorescence intensity before and after the treatment were measured.The results are shown in FIG. 7. The reactive oxygen species andreactive nitrogen species were prepared as follows:

-   -   a. H₂O₂ (final 100 μM) was added and then stirred for 1 hour at        25° C.    -   b. (3-(1,4-Dihydro-1,4-epidioxy-1-naphthyl)propionic acid)        (final 100 μM) was added and then stirred at 25° C. for 1 hour.    -   c. 2,2′-Azobis(2-amidinopropane)dihydrochloride (final 100 μM)        was added and then stirred at 25° C. for 1 hour.    -   d. SNP (Sodium Nitroferricyanide (III) Dihydrate) (final 100 μM)        was added and then stirred for 1 hour at 25° C.    -   e. O₂ ^(•−) was generated by xanthine and xanthine oxidase.        Xanthine oxidase was added firstly. After xanthine oxidase was        dissolved, xanthine (final 100 μM) was added and the mixtures        were stirred at 25° C. for 1 hour.    -   f. Ferrous chloride (final 10 μM) was added in the presence of        10 equivalences of H₂O₂ (100 μM).    -   g. ONOO⁻ (final 10 μM) was added at 25° C. Peroxynitrite was        prepared as stated in Example 4.    -   h. NaOCl (final 10 μM) was added at 25° C.

FIG. 7 shows that hypochlorite leads to much stronger fluorescenceenhancement of Compound 14 than any other ROS and RNS. These resultsdemonstrated that Compound 14 has a much higher reactivity towardshypochlorite among ROS and RNS in an abiotic system. Further, similarreactions do not proceed between the aniline derivative of Compound 14and any other reactive oxygen species or reactive nitrogen speciespresent in the biological systems.

Example 12 Application of Compounds 12 and 12a in Cell Assay

Murine J744.1 macrophages (ATCC, USA) were used to investigate thepotential of Compound 12 and 12a for the detection of peroxynitrite inliving cells. J744.1 macrophages were cultured in Dulbecco's ModifiedEagle's Medium (DMEM) (Gibco) containing 10% heat-inactivated fetalbovine serum (Gibco) supplemented with 100 U/ml of penicillin and 100μg/ml streptomycin at 37° C., 5% CO₂. They were subcultured by scrapingand seeded on six-well plates according to manufacture's instruction.The growth medium was changed every two to three days. Cells were grownto confluence prior to experiment. Murine J744.1 macrophages wereincubated with Compound 12 or 12a (20 μM) for 1 hr and then washed threetimes with PBS buffer. Only very weak fluorescence was observed in theabsence of stimulants (FIG. 9B and FIG. 10A). The fluorescence wasinduced after treatment with LPS (lipopolysaccharide, 1 μg/ml) for 4 hrs(FIG. 9D and FIG. 10B). Also the green color from Compound 12 was foundto be colocalized with the red color from a mitochondrial dyeMitoTracker Red CMXRos (FIG. 9F). The results indicate that Compound 12may selectively localize in mitochondrials.

Example 13 Detection of Peroxynitrite with Compound 30

UV-Visible Absorption Spectrum of Compound 30

Compound 30 obtained in Example 7 was dissolved in pH 7.4 0.1 Mphosphate buffer containing 0.1% DMF as cosolvent to form a 10 μMsolution. The absorption spectrum of the 10 μM solution of Compound 30was measured. The absorption maximum of Compound 30 was found to be atabout 540 nm.

Emission Spectra of Compound 30

Compound 30 obtained in Example 7 was dissolved in DMF to aconcentration of 10 mM, and then the solution was diluted to 10 μM by0.1 M phosphate buffer (pH 7.4). The fluorescence spectrum of the 10 μMsolution of Compound 30 was measured using a Hitachi F7000 fluorescencespectrometer and the photomultiplier voltage was 900 V. The slit widthwas 2.5 nm for both excitation and emission. The measurement was carriedout at an excitation wavelength of 520 nm. The results shown in FIG. 11indicate that Compound 30 itself may be non-fluorescent.

Detection of Peroxynitrite with Compound 30

Compound 30 obtained in Example 7 was dissolved in DMF to aconcentration of 10 mM, and then the solution was diluted to 10 μM by0.1 M potassium phosphate buffer (pH 7.4). Peroxynitrite solution in 0.1M NaOH was prepared by the method of Keith and Powell (Keith, W. G. &Powell, R. E.; Kinetics of decomposition of peroxynitrous acid; J. Chem.Soc. A, 1969, 1, 90), and its concentration in the stock solutions usedwas estimated by using an extinction coefficient of 1670 cm⁻¹ (mol/L)⁻¹at 302 nm (Hughes and Nicklin; The chemistry of pernitrites. Part I.Kinetics of decomposition of pernitrious acid; J. Chem. Soc. A, 1968, 2,450-452). Peroxynitrite stock solution was added into the solution ofCompound 30 to provide various final concentrations. Fluorescencespectra of the solutions were measured after 5 minutes under the sameconditions as mentioned above. The fluorescence spectra are shown inFIG. 11. As clearly shown in FIG. 11, the fluorescence intensity ofCompound 30 increases significantly after the addition of peroxynitrite.

As demonstrated above, embodiments disclosed herein provide variouscompounds that can be used as fluorogenic probes for detecting,measuring and/or screening peroxynitrite. While this disclosure has beendescribed with respect to a limited number of embodiments, the specificfeatures of one embodiment should not be attributed to otherembodiments. No single embodiment is representative of all aspects ofthis disclosure. In some embodiments, the compositions or methods mayinclude numerous compounds or steps not mentioned herein. In otherembodiments, the compositions or methods do not include, or aresubstantially free of, any compounds or steps not enumerated herein.Variations and modifications from the described embodiments exist. Forexample, the reagent composition disclosed herein need not comprisingonly the fluorogenic probes disclosed herein. It can comprise any typeof compounds generally suitable for fluorogenic probes. It is noted thatthe methods for making and using the fluorogenic probes disclosed hereinare described with reference to a number of steps. These steps can bepracticed in any sequence. One or more steps may be omitted or combinedbut still achieve substantially the same results. The appended claimsintend to cover all such variations and modifications as falling withinthe scope of this disclosure.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. It is to beunderstood that this disclosure has been described in detailed by way ofillustration and example in order to acquaint others skilled in the artwith the invention, its principles, and its practical application.Further, the specific embodiments provided herein as set forth are notintended to be exhaustive or to limit the disclosure, and that manyalternatives, modifications, and variations will be apparent to thoseskilled in the art in light of the foregoing examples and detaileddescription. Accordingly, this disclosure is intended to embrace allsuch alternatives, modifications, and variations that fall within thespirit and scope of the following claims. While some of the examples anddescriptions above include some conclusions about the way the compounds,compositions and methods may function, the inventors do not intend to bebound by those conclusions and functions, but put them forth only aspossible explanations in light of current understanding.

What is claimed is:
 1. An aromatic amine compound of Formula (I):

wherein R¹ is hydrogen, alkyl, halogenated alkyl, heteroalkyl, alkenyl,alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenylor cycloalkynyl; L is formula (II):

 or a tautomer thereof, wherein Y is O-A, S-A or NR²R³; each of R² andR³ is independently H, alkyl, halogenated alkyl, alkenyl, alkynyl,alkoxyalkyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,heterocyclyl, aminoalkyl, aryl, alkaryl, arylalkyl, alkyloxy,carboxyalkyl, alkylamido, alkoxyamido, sulfonylaryl or acyl; A is H,alkyl, alkenyl, alkynyl, alkoxyalkyl, heteroalkyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heterocyclyl, aminoalkyl, aryl, alkaryl,arylalkyl, carboxyalkyl, alkoxycarbonyl, acyl or aminocarbonyl; each ofK¹-K¹⁰ is independently H, halo, alkyl, halogenated alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, alkaryl, heterocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino,arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino,hydroxy, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy,arylalkoxy, acyloxy, cyano, nitro, sulfhydryl, carbamoyl,trifluoromethyl, phenoxy, benzyloxy, sulfonyl, phosphonyl, sulfonateester, phosphate ester, —C(═O)—P¹ or —C(═O)—Z—P²; each of P¹ and P² isindependently hydrogen, halo, alkoxy, hydroxy, thiol, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl,arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino,alkylarylamino, diarylamino, alkylthio, heteroalkyl, or heterocyclylhaving from 3 to 7 ring atoms; and Z is alkylene, alkynylene,alkynylene, arylene, aralkylene or alkarylene; and Q is substituted orunsubstituted phenyl having formula (VIIa):

wherein each of R⁴, R⁵, R⁶, R⁷ and R⁸ is independently H, alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,aryl, alkylaryl, arylalkyl, heterocyclyl, hydroxy, alkoxy, alkoxyalkyl,alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogenated alkylcarbonylalkyl,aminoalkyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl,aminocarbonyl, or NR⁹R¹⁰ or R⁴ and R⁵ together, R⁵ and R⁶ together, R⁶and R⁷ together or R⁷ and R⁸ together forming a 5- or 6-memberedcycloalkyl, heterocyclyl, aryl orheteroaryl ring fused with the phenylring of formula (VIIa); and each of R⁹ and R¹⁰ is independently H,alkyl, alkenyl, alkynyl, alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl,aryloyl, or polyether; with the proviso that when L has formula (II)where Y is NR²R³, then R⁶ of Q is hydroxy, alkenyl, alkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclyl,alkoxyalkyl, alkoxyalkoxy, acyl, alkylcarbonylalkyl, halogenatedalkylcarbonylalkyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl oraminocarbonyl, or R⁴ and R⁵ together, R⁵ and R⁶ together, R⁶ and R⁷together or R⁷ and R⁸ together form a 5- or 6-membered cycloalkyl,heterocyclyl, aryl or heteroaryl ring fused with the phenyl ring offormula (VIIa).
 2. The aromatic amine compound of claim 1, wherein R⁶ is—OCH₂OCH₃, OH, NR⁹R¹⁰, —CH₂CH₂C(═O)CF₃, or —CH₂CH₂C(═O)OCH₃ where eachof R⁹ and R¹⁰ is independently H or alkyl; and each of R⁴, R⁵, R⁷ and R⁸is H.
 3. The aromatic amine compound of claim 2, wherein R⁶ is OH, NH₂or —CH₂CH₂C(═O)CF₃.
 4. The aromatic amine compound of claim 1, whereinR¹ is H, alkyl, halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aryl,aralkyl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, andcycloalkynyl; each of R⁴, R⁵, R⁶, R⁷ and R⁸ is independently H, halogen,alkyl, alkoxy, or polyether; R⁶ is OR¹¹ or CH₂CH₂COR¹², where R¹¹ is H,alkyl, alkoxyalkyl, alkanoyl, or polyether; R¹² is anelectron-withdrawing group selected from CF₃ , halogen-substituted loweralkyl, or (C═O)—O—V²; and V² is a group selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl orarylalkyl.
 5. The aromatic amine compound of claim 1, wherein L has oneof the following formulae:

or a tautomer thereof, wherein each of said formulae is independentlyunsubstituted or substituted.
 6. The aromatic amine compound of claim 1,wherein the aromatic amine compound is one of the following Compounds:

or a tautomer thereof, wherein each of said Compounds is independentlysubstituted or unsubstituted.
 7. A composition comprising the aromaticamine compound of claim 1 and a carrier.
 8. The composition of claim 7,wherein the aromatic amine compound is Compound (10), Compound (12),Compound (12a), or Compound (22):

or a tautomer thereof, or a combination thereof.
 9. The composition ofclaim 7, wherein the composition further comprises a solvent, an acid, abase, a buffer solution or a combination thereof.
 10. The composition ofclaim 7, wherein the aromatic amine compound is Compound (14):

or a tautomer thereof.